Clinical Chemistry: Principles, Techniques, and Correlations (8th Edition) PDF

EIGHTH edition Clinical Chemistry Principles, Techniques, and Correlations EIGHTH edition Clinical Chemistry Principles, Techniques, and...

EIGHTH edition Clinical Chemistry Principles, Techniques, and Correlations EIGHTH edition Clinical Chemistry Principles, Techniques, and Correlations Michael L. Bishop, MS, MLS(ASCP)CM Campus Department Chair Medical Laboratory Science Keiser University Orlando, Florida Edward P. Fody, MD Clinical Professor Department of Pathology, Microbiology and Immunology Vanderbilt University School of Medicine Nashville, Tennessee Medical Director Department of Pathology Holland Hospital Holland, Michigan Larry E. Schoeff, MS, MT(ASCP) Professor (Retired), Medical Laboratory Science Program Department of Pathology University of Utah School of Medicine Salt Lake City, Utah Acquisitions Editor: Jonathan Joyce Product Development Editor: John Larkin Marketing Manager: Leah Thomson Production Project Manager: Kim Cox Design Coordinator: Joan Wendt Manufacturing Coordinator: Margie Orzech Prepress Vendor: SPi Global Eighth edition Copyright © 2018 Wolters Kluwer Copyright © 2013, 2010, 2005, 2000, 1996, 1992, 1985 Wolters Kluwer Health/Lippincott Williams & Wilkins. All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Wolters Kluwer at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, via email at [email protected], or via our website at lww.com (products and services). 987654321 Printed in China Library of Congress Cataloging-in-Publication Data Names: Bishop, Michael L., editor. | Fody, Edward P., editor. | Schoeff, Larry E., editor. Title: Clinical chemistry : principles, techniques, and correlations / [edited by] Michael L. Bishop, Edward P. Fody, Larry E. Schoeff. Other titles: Clinical chemistry (Bishop) Description: Eighth edition. | Philadelphia : Wolters Kluwer, | Includes bibliographical references and index. Identifiers: LCCN 2016031591 | ISBN 9781496335586 Subjects: | MESH: Clinical Chemistry Tests | Chemistry, Clinical—methods Classification: LCC RB40 | NLM QY 90 | DDC 616.07/56—dc23 LC record available at https://lccn.loc.gov/2016031591 This work is provided “as is,” and the publisher disclaims any and all warranties, express or implied, including any warranties as to accuracy, comprehensiveness, or currency of the content of this work. This work is no substitute for individual patient assessment based upon healthcare professionals' examination of each patient and consideration of, among other things, age, weight, gender, current or prior medical conditions, medication history, laboratory data and other factors unique to the patient. The publisher does not provide medical advice or guidance and this work is merely a reference tool. Healthcare professionals, and not the publisher, are solely responsible for the use of this work including all medical judgments and for any resulting diagnosis and treatments. Given continuous, rapid advances in medical science and health information, independent professional verification of medical diagnoses, indications, appropriate pharmaceutical selections and dosages, and treatment options should be made and healthcare professionals should consult a variety of sources. When prescribing medication, healthcare professionals are advised to consult the product information sheet (the manufacturer's package insert) accompanying each drug to verify, among other things, conditions of use, warnings and side effects and identify any changes in dosage schedule or contraindications, particularly if the medication to be administered is new, infrequently used or has a narrow therapeutic range. To the maximum extent permitted under applicable law, no responsibility is assumed by the publisher for any injury and/or damage to persons or property, as a matter of products liability, negligence law or otherwise, or from any reference to or use by any person of this work. LWW.com Not authorised for sale in United States, Canada, Australia, New Zealand, Puerto Rico, and U.S. Virgin Islands. Acquisitions Editor: Jonathan Joyce Product Development Editor: John Larkin Marketing Manager: Leah Thomson Production Project Manager: Kim Cox Design Coordinator: Joan Wendt Manufacturing Coordinator: Margie Orzech Prepress Vendor: SPi Global Eighth edition Copyright © 2018 Wolters Kluwer Copyright © 2013, 2010, 2005, 2000, 1996, 1992, 1985 Wolters Kluwer Health/Lippincott Williams & Wilkins. All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Wolters Kluwer at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, via email at [email protected], or via our website at lww.com (products and services). 987654321 Printed in China Library of Congress Cataloging-in-Publication Data Names: Bishop, Michael L., editor. | Fody, Edward P., editor. | Schoeff, Larry E., editor. Title: Clinical chemistry : principles, techniques, and correlations / [edited by] Michael L. Bishop, Edward P. Fody, Larry E. Schoeff. Other titles: Clinical chemistry (Bishop) Description: Eighth edition. | Philadelphia : Wolters Kluwer, | Includes bibliographical references and index. Identifiers: LCCN 2016031591 | ISBN 9781496365392 Subjects: | MESH: Clinical Chemistry Tests | Chemistry, Clinical—methods Classification: LCC RB40 | NLM QY 90 | DDC 616.07/56—dc23 LC record available at https://lccn.loc.gov/2016031591 This work is provided “as is,” and the publisher disclaims any and all warranties, express or implied, including any warranties as to accuracy, comprehensiveness, or currency of the content of this work. This work is no substitute for individual patient assessment based upon healthcare professionals' examination of each patient and consideration of, among other things, age, weight, gender, current or prior medical conditions, medication history, laboratory data and other factors unique to the patient. The publisher does not provide medical advice or guidance and this work is merely a reference tool. Healthcare professionals, and not the publisher, are solely responsible for the use of this work including all medical judgments and for any resulting diagnosis and treatments. Given continuous, rapid advances in medical science and health information, independent professional verification of medical diagnoses, indications, appropriate pharmaceutical selections and dosages, and treatment options should be made and healthcare professionals should consult a variety of sources. When prescribing medication, healthcare professionals are advised to consult the product information sheet (the manufacturer's package insert) accompanying each drug to verify, among other things, conditions of use, warnings and side effects and identify any changes in dosage schedule or contraindications, particularly if the medication to be administered is new, infrequently used or has a narrow therapeutic range. To the maximum extent permitted under applicable law, no responsibility is assumed by the publisher for any injury and/or damage to persons or property, as a matter of products liability, negligence law or otherwise, or from any reference to or use by any person of this work. LWW.com In memory of my mother, Betty Beck Bishop, for her constant support, guidance, and encouragement. MLB To Nancy, my wife, for continuing support and dedication. EPF To my wife, Anita, for continuing support. LES  Contributors Dev Abraham, MD Professor of Medicine Division of Endocrinology University of Utah Salt Lake City, Utah Josephine Abraham, MD, MPH Professor of Medicine Division of Nephrology University of Utah Salt Lake City, Utah Michael J. Bennett, PhD, FRCPath, FACB, DABCC Professor of Pathology and Laboratory Medicine University of Pennsylvania Director, Metabolic Disease Laboratory Children's Hospital of Philadelphia Abramson Pediatric Research Center Philadelphia, Pennsylvania Takara L. Blamires, M.S., MLS(ASCP)CM Medical Laboratory Science Program Department of Pathology University of Utah School of Medicine Salt Lake City, Utah Maria G. Boosalis, PhD, MPH, RD, LD Professor, College of Health and Wellness Northwestern Health Sciences University Bloomington, Minnesota Raffick A. R. Bowen, PhD, MHA, MT(CSMLS), DClChem, FCACB, DABCC, FACB Clinical Associate Professor of Pathology Associate Director of Clinical Chemistry and Immunology Laboratory Department of Pathology Stanford Health Care Stanford, California Janelle M. Chiasera, PhD Department Chair, Clinical and Diagnostic Sciences University of Alabama – Birmingham Birmingham, Alabama Heather Corn, MD Internal Medicine – Clinical Instructor Endocrinology and Diabetes Center University of Utah Salt Lake City, Utah Heather Crookston, PhD Point of Care Coordinator Nemours Children's Hospital Orlando, Florida Julia C. Drees, PhD, DABCC Clinical Research and Development Scientist Kaiser Permanente Regional Laboratory Berkeley, California Kathryn Dugan, MEd, MT(ASCP) Instructor Medical and Clinical Laboratory Sciences Auburn University at Montgomery Montgomery, Alabama Michael Durando, MD, PhD Research Track Resident, Internal Medicine and Hematology/Oncology Emory University Atlanta, Georgia Edward P. Fody, MD Clinical Professor Department of Pathology, Microbiology and Immunology Vanderbilt University School of Medicine Nashville, Tennessee Medical Director Department of Pathology Holland Hospital Holland, Michigan Elizabeth L. Frank, PhD Associate Professor, Department of Pathology University of Utah School of Medicine Medical Director, Analytic Biochemistry and Calculi ARUP Laboratories, Inc. Salt Lake City, Utah Vicki S. Freeman, PhD, MLS(ASCP)CM SC, FACB Department Chair and Associate Professor, Clinical Laboratory Sciences University of Texas Medical Branch Galveston, Texas Linda S. Gorman, PhD Retired Associate Professor Medical Laboratory Science University of Kentucky Lexington, Kentucky Ryan W. Greer, MS, I&C(ASCP) Assistant Vice President, Group Manager Chemistry Group III, Technical Operations ARUP Laboratories, Inc. Salt Lake City, Utah Marissa Grotzke, MD Assistant Professor Endocrinology and Metabolism Internal Medicine University of Utah Salt Lake City, Utah Mahima Gulati, MD Endocrinologist Middlesex Hospital Danbury, Connecticut Carrie J. Haglock-Adler, MSFS, C(ASCP) Research Scientist ARUP Institute for Clinical and Experimental Pathology Salt Lake City, Utah Matthew P. A. Henderson, PhD, FCACB Clinical Biochemist and Laboratory Director for the Children's Hospital of Eastern Ontario Ottawa Hospital Assistant Professor Department of Pathology and Laboratory Medicine at the University of Ottawa University of Ottawa Ottawa, Ontario, Canada Ronald R. Henriquez, PhD, NRCC Fellow, Clinical Chemistry University of North Carolina at Chapel Hill, Pathology and Laboratory Medicine University of North Carolina Chapel Hill, North Carolina Clinical Chemist, Department of Pathology Walter Reed National Military Medical Center, United States Army Bethesda, Maryland Laura M. Hickes, PhD Chemistry and Applications Support Manager Roche Diagnostics Greensboro, North Carolina Brian C. Jensen, MD Assistant Professor of Medicine and Pharmacology UNC Division of Cardiology UNC McAllister Heart Institute University of North Carolina Chapel Hill, North Carolina Kamisha L. Johnson-Davis, PhD, DABCC (CC, TC), FACB Assistant Professor (Clinical) Department of Pathology University of Utah Medical Director, Clinical Toxicology ARUP Laboratories Salt Lake City, Utah Robert E. Jones, MD Professor of Medicine Endocrinology and Diabetes Center Division of Endocrinology University of Utah Salt Lake City, Utah Yachana Kataria, Phd Clinical Chemistry Fellow Department of Laboratory Medicine Boston Children's Hospital Research Fellow Harvard Medical School Boston, Massachusetts Mark D. Kellogg, PhD, MT(ASCP), DABCC, FACB Director of Quality Programs Associate Director of Chemistry Department of Laboratory Medicine Boston Children's Hospital Assistant Professor of Pathology Harvard Medical School Boston, Massachusetts Cindi Bullock Letsos, MT(ASCP) Lean Six Sigma Black Belt Consultant Retired from University of North Carolina Health Care Chapel Hill, North Carolina Kara L. Lynch, PhD Associate Division Chief, Chemistry and Toxicology Laboratory San Francisco General Hospital San Francisco, California J. Marvin McBride, MD, MBA Assistant Clinical Professor Division of Geriatric Medicine UNC School of Medicine Chapel Hill, North Carolina Christoper R. McCudden, PhD Clinical Biochemist, Pathology and Laboratory Medicine The Ottawa Hospital Assistant Professor Department of Pathology and Laboratory Medicine at the University of Ottawa University of Ottawa Ottawa, Ontario, Canada Shashi Mehta, PhD Associate Professor Department of Clinical Laboratory Sciences School of Health Related Professions University of Medicine and Dentistry of New Jersey Newark, New Jersey James March Mistler, MS, MLS Lecturer Department of Medical Laboratory Science University of Massachusetts Dartmouth North Dartmouth, Massachusetts Matthew S. Petrie, PhD Clinical Chemistry Fellow, Department of Laboratory Medicine University of California San Francisco San Francisco, California Tracey G. Polsky, MD, PhD Assistant Professor of Clinical Pathology and Laboratory Medicine University of Pennsylvania Perelman School of Medicine Assistant Director of the Clinical Chemistry Laboratory Children's Hospital of Philadelphia Philadelphia, Pennsylvania Deepika S. Reddy, MD Assistant Professor (Clinical) Internal Medicine Endocrinology and Diabetes Center University of Utah Salt Lake City, Utah Alan T. Remaley, MD, PhD Senior Staff, Department of Laboratory Medicine National Institutes of Health Bethesda, Maryland Kyle B. Riding, PhD, MLS(ASCP) Instructor, Medical Laboratory Science Teaching and Learning Center Coordinator Keiser University – Orlando Campus Orlando, Florida Michael W. Rogers, MT(ASCP), MBA Clinical Laboratory Quality Management Consultant Retired from University of North Carolina Health Care Chapel Hill, North Carolina Amar A. Sethi, PhD Chief Scientific Officer, Research and Development Pacific Biomarkers Seattle, Washington Joely A. Straseski, PhD Assistant Professor, Pathology University of Utah Salt Lake City, Utah Frederick G. Strathmann, PhD, DABCC (CC, TC) Assistant Professor Department of Pathology University of Utah Medical Director of Toxicology ARUP Laboratories Salt Lake City, Utah Vishnu Sundaresh, MD, CCD Assistant Professor (Clinical) Internal Medicine Endocrinology and Diabetes Center University of Utah Salt Lake City, Utah Sara A. Taylor, PhD, MLS(ASCP), MB Associate Professor and Graduate Advisor Department of Medical Laboratory Science Tarleton State University Fort Worth, Texas Tolmie E. Wachter, MBA/HCM, SLS(ASCP) Assistant Vice President Director of Corporate Safety/RSO ARUP Laboratories Salt Lake City, Utah G. Russell Warnick, MS, MBA Chief Scientific Officer Health Diagnostic Laboratory Richmond, Virginia Elizabeth Warning, MS, MLS(ASCP)CM Adjunct Faculty MLS Program University of Cincinnati Cincinnati, Ohio Monte S. Willis, MD, PhD, FCAP, FASCP, FAHA Associate Professor, Vice Chair of Academic Affairs Department of Pathology & Laboratory Medicine, Director, Campus Health Services Laboratory University of North Carolina at Chapel Hill, Pathology & Laboratory Medicine Director, Sweat Chloride Testing Assistant Director Clinical Core (Chemistry) Laboratory Services University of North Carolina Healthcare University of North Carolina Chapel Hill, North Carolina Alan H. B. Wu, PhD, DABCC Director, Clinical Chemistry Laboratory, San Francisco General Hospital Professor, Laboratory Medicine, University of California, San Francisco San Francisco, California Xin Xu, MD, PhD, MLS(ASCP) Division of Pulmonary, Allergy, and Critical Care MedicineDepartment of Medicine University of Alabama at Birmingham Birmingham, AL  foreword to the eighth edition For many years, the health care and medical laboratory communities have been preparing for an impending workforce shortage that could threaten to compromise patient care and safety. It is vital that the medical laboratory community continue to educate and prepare credentialed professionals who can work efficiently, have essential analytical and critical thinking skills, and can communicate test results and test needs to health care providers. While the shortage of qualified laboratory practitioners has been in the forefront of our collective thoughts, a more insidious shortage has also arisen among the ranks of faculty within medical laboratory education programs. As a profession, we have long been blessed with dedicated faculty who strive to impart their knowledge and experience onto countless students. We know, however, that many of these dedicated faculty members have and will continue to step aside to pursue other passions through retirement. As new dedicated faculty step up to take over, we must support these new educators in their roles as program directors and content specialists. Furthermore, we must provide tools for these educators about techniques and theories that are appropriate as they continue to develop their curricula. One potential tool to assist educators is the American Society for Clinical Laboratory Science (ASCLS) Entry Level Curriculum. At the 2016 ASCLS Annual Meeting, the House of Delegates adopted a newly formatted version of the entry level curriculum for MLT and MLS programs. This document had not been updated since 2002, and a subcommittee of the ASCLS Education Scientific Assembly was charged with editing the document to better represent the field's expectations of new graduates. Subcommittee members solicited feedback from educators and professionals in all subdisciplines and reviewed the content for currency. New material was added to reflect techniques and theories that have emerged since the last edition while material was removed if it was no longer deemed relevant. After this extensive process, the final document is reflective of what the industry demands of a new professional. Similarly, the material presented throughout Clinical Chemistry: Principles, Techniques, and Correlations has always kept current with changes in the laboratory industry. This exceptional ability of authors and editors to keep pace with the needs of an ever-changing profession has not diminished through its, now, eight editions. The content inherent to the discipline of clinical chemistry is foundational to all other areas of laboratory medicine. The eighth edition of this textbook is ideal for students learning the principles of clinical chemistry while helping them build connections to other areas of the laboratory. The chapters have a perfect blend of basic theory and practical information that allows the student to comprehend each area of clinical chemistry. The text is well organized to help MLT and MLS educators distinguish what each unique student population needs to be successful in the marketplace. The online materials, powerpoints, and exam questions, for educators, are an invaluable resource for those creating a new course or revising a current course. As we face this transition of laboratory practitioners who perform testing and faculty who train and educate our students, products that stay current with the times and help facilitate better understanding of the unique levels of practice within our field are the most essential element to success. The eighth edition of Clinical Chemistry: Principles, Techniques, and Correlations accomplishes this and serves as an invaluable tool for any new educator looking for guidance, or seasoned educator looking to refresh their teachings. As educators we are thrilled that students continue to find the field of medical laboratory science an avenue to build a professional career. We wish all students and educators who use this book the best to carry on a tradition of excellence! Joan Polancic, MSEd, MLS(ASCP)CM Director, School of Medical Laboratory Science Denver Health Medical Center Denver, Colorado Kyle B. Riding, PhD, MLS(ASCP) Instructor, Medical Laboratory Science Teaching and Learning Center Coordinator Keiser University – Orlando Campus Orlando, Florida  foreword to the seventh edition You should not be surprised to learn that the delivery of health care has been undergoing major transformation for several decades. The clinical laboratory has been transformed in innumerable ways as well. At one time, the laboratory students' greatest asset was motor ability. That is not the case any longer. Now the need is for a laboratory professional who is well educated, an analytical thinker, and problem solver, and one who can add value to the information generated in the laboratory regarding a specific patient. This change impacts the laboratory professional in a very positive manner. Today the students' greatest asset is their mental skill and their ability to acquire and apply knowledge. The laboratory professional is now considered a knowledge worker, and a student's ability to successfully become this knowledge worker depends on their instruction and exposure to quality education. Herein lies the need for the seventh edition of Clinical Chemistry: Principles, Techniques, and Correlations. It contributes to the indispensable solid science foundation in medical laboratory sciences and the application of its principles in improving patient outcomes needed by the laboratory professional of today. This edition provides not only a comprehensive understanding of clinical chemistry but also the foundation upon which all the other major laboratory science disciplines can be further understood and integrated. It does so by providing a strong discussion of organ function and a solid emphasis on pathophysiology, clinical correlations, and differential diagnosis. This information offers a springboard to better understand the many concepts related to the effectiveness of a particular test for a particular patient. Reduction of health care costs, while ensuring quality patient care, remains the goal of health care reform efforts. Laboratory information is a critical element of such care. It is estimated that $65 billion is spent each year to perform more than 4.3 billion laboratory tests. This impressive figure has also focused a bright light on laboratory medicine, and appropriate laboratory test utilization is now under major scrutiny. The main emphasis is on reducing costly overutilization and unnecessary diagnostic testing; however, the issue of under- and misutilization of laboratory tests must be a cause for concern as well. The role of laboratorians in providing guidance to clinicians regarding appropriate test utilization is becoming not only accepted but also welcomed as clinicians try to maneuver their way through an increasingly complex and expensive test menu. These new roles lie in the pre- and postanalytic functions of laboratorians. The authors of this text have successfully described the importance of these phases as well as the more traditional analytic phase. It does not matter how precise or accurate a test is during the analytic phase if the sample has been compromised or if an inappropriate test has been ordered on the patient. In addition, the validation of results with respect to a patient's condition is an important step in the postanalytic phase. Participation with other health care providers in the proper interpretation of test results and appropriate follow-up will be important abilities of future graduates as the profession moves into providing greater consultative services for a patient-centered medical delivery system. Understanding these principles is a necessary requirement of the knowledge worker in the clinical laboratory. This significant professional role provides effective laboratory services that will improve medical decision making and thus patient safety while reducing medical errors. This edition of Clinical Chemistry: Principles, Techniques, and Correlations is a crucial element in graduating such professionals. Diana Mass, MA, MT(ASCP) Clinical Professor and Director (Retired) Clinical Laboratory Sciences Program Arizona State University Tempe, Arizona President Associated Laboratory Consultants Valley Center, California Make no mistake: There are few specialties in medicine that have a wider impact on today's health care than laboratory medicine. For example, in the emergency room, a troponin result can not only tell an ER physician if a patient with chest pain has had a heart attack but also assess the likelihood of that patient suffering an acute myocardial infarction in 30 days. In the operating room during a parathyroidectomy, a parathyroid hormone assay can tell a surgeon that it is appropriate to close the procedure because he has successfully removed all of the affected glands or go back and look for more glands to excise. In labor and delivery, testing for pulmonary surfactants from amniotic fluid can tell an obstetrician if a child can be safely delivered or if the infant is likely to develop life-threatening respiratory distress syndrome. In the neonatal intensive care unit, measurement of bilirubin in a premature infant is used to determine when the ultraviolet lights can be turned off. These are just a handful of the thousands of medical decisions that are made each day based on results from clinical laboratory testing. Despite our current success, there is still much more to learn and do. For example, there are no good laboratory tests for the diagnosis of stroke or traumatic brain injury. The work on Alzheimer's and Parkinson's disease prediction and treatment is in the early stages. And when it comes to cancer, while our laboratory tests are good for monitoring therapy, they fail in the detection of early cancer, essential for improving treatment and prolonging survival. Finally, personalized medicine including pharmacogenomics will play an increasingly important role in the future. Pharmacogenomic testing will be used to select the right drug at the best dose for a particular patient in order to maximize efficacy and minimize side effects. If you are reading this book, you are probably studying to be a part of the field. As a clinical chemist for over 30 years, I welcome you to our profession.  Alan H. B. Wu, PhD, DABCC Director, Clinical Chemistry Laboratory, San Francisco General Hospital Professor, Laboratory Medicine, University of California, San Francisco San Francisco, California  Preface Clinical chemistry continues to be one of the most rapidly advancing areas of laboratory medicine. Since the initial idea for this textbook was discussed in a meeting of the Biochemistry/Urinalysis section of ASMT (now ASCLS) in the late 1970s, the only constant has been changed. New technologies and analytical techniques have been introduced, with a dramatic impact on the practice of clinical chemistry and laboratory medicine. In addition, the health care system is rapidly changing. There is ever increasing emphasis on improving the quality of patient care, individual patient outcomes, financial responsibility, and total quality management. Now, more than ever, clinical laboratorians need to be concerned with disease correlations, interpretations, problem solving, quality assurance, and cost-effectiveness; they need to know not only the how of tests but more importantly the what, why, and when. The editors of Clinical Chemistry: Principles, Techniques, and Correlations have designed the eighth edition to be an even more valuable resource to both students and practitioners. Now almost 40 years since the initiation of this effort, the editors have had the privilege of completing the eighth edition with another diverse team of dedicated clinical laboratory professionals. In this era of focusing on metrics, the editors would like to share the following information. The 330 contributors in the 8 editions represent 70 clinical laboratory science programs, 83 clinical laboratories, 13 medical device companies, 4 government agencies, and 3 professional societies. One hundred and thirty contributors were clinical laboratory scientists with advanced degrees. With today's global focus, the previous editions of the text have been translated into at least six languages. By definition, a profession is a calling requiring specialized knowledge and intensive academic preparation to define its scope of work and produce its own literature. The profession of Clinical Laboratory Science has evolved significantly over the past four decades. The eighth edition of Clinical Chemistry: Principles, Techniques, and Correlations is comprehensive, up-to-date, and easy to understand for students at all levels. It is also intended to be a practically organized resource for both instructors and practitioners. The editors have tried to maintain the book's readability and further improve its content. Because clinical laboratorians use their interpretative and analytic skills in the daily practice of clinical chemistry, an effort has been made to maintain an appropriate balance between analytic principles, techniques, and the correlation of results with disease states. In this edition, the editors have maintained features in response to requests from our readers, students, instructors, and practitioners. Ancillary materials have been updated and expanded. Chapters now include current, more frequently encountered case studies and practice questions or exercises. To provide a thorough, up-to-date study of clinical chemistry, all chapters have been updated and reviewed by professionals who practice clinical chemistry and laboratory medicine on a daily basis. The basic principles of the analytic procedures discussed in the chapters reflect the most recent or commonly performed techniques in the clinical chemistry laboratory. Detailed procedures have been omitted because of the variety of equipment and commercial kits used in today's clinical laboratories. Instrument manuals and kit package inserts are the most reliable reference for detailed instructions on current analytic procedures. All chapter material has been updated, improved, and rearranged for better continuity and readability. thePoint*, a Web site with additional case studies, review questions, teaching resources, teaching tips, additional references, and teaching aids for instructors and students, is available from the publisher to assist in the use of this textbook. Michael L. Bishop Edward P. Fody Larry E. Schoeff  Acknowledgments A project as large as this requires the assistance and support of many clinical laboratorians. The editors wish to express their appreciation to the contributors of all the editions of Clinical Chemistry: Principles, Techniques, and Correlations—the dedicated laboratory professionals and educators whom the editors have had the privilege of knowing and exchanging ideas with over the years. These individuals were selected because of their expertise in particular areas and their commitment to the education of clinical laboratorians. Many have spent their professional careers in the clinical laboratory, at the bench, teaching students, or consulting with clinicians. In these frontline positions, they have developed a perspective of what is important for the next generation of clinical laboratorians. We extend appreciation to our students, colleagues, teachers, and mentors in the profession who have helped shape our ideas about clinical chemistry practice and education. Also, we want to thank the many companies and professional organizations that provided product information and photographs or granted permission to reproduce diagrams and tables from their publications. Many Clinical and Laboratory Standards Institute (CLSI) documents have also been important sources of information. These documents are directly referenced in the appropriate chapters. The editors would like to acknowledge the contribution and effort of all individuals to previous editions. Their efforts provided the framework for many of the current chapters. Finally, we gratefully acknowledge the cooperation and assistance of the staff at Wolters Kluwer for their advice and support. The editors are continually striving to improve future editions of this book. We again request and welcome our readers' comments, criticisms, and ideas for improvement.  Contents Contributors Foreword to the Eighth Edition Foreword to the Seventh Edition Preface Acknowledgments PART one Basic Principles and Practice of Clinical Chemistry 1 Basic Principles and Practices Kathryn Dugan and Elizabeth Warning UNITS OF MEASURE REAGENTS Chemicals Reference Materials Water Specifications Solution Properties Concentration Colligative Properties Redox Potential Conductivity pH and Buffers CLINICAL LABORATORY SUPPLIES Thermometers/Temperature Glassware and Plasticware  Desiccators and Desiccants Balances CENTRIFUGATION LABORATORY MATHEMATICS AND CALCULATIONS Significant Figures Logarithms Concentration Dilutions Water of Hydration Graphing and Beer's Law SPECIMEN CONSIDERATIONS Types of Samples Sample Processing Sample Variables Chain of Custody Electronic and Paper Reporting of Results QUESTIONS REFERENCES 2 Laboratory Safety and Regulations Tolmie E. Wachter LABORATORY SAFETY AND REGULATIONS Occupational Safety and Health Act Other Regulations and Guidelines SAFETY AWARENESS FOR CLINICAL LABORATORY PERSONNEL Safety Responsibility Signage and Labeling SAFETY EQUIPMENT Chemical Fume Hoods and Biosafety Cabinets Chemical Storage Equipment PPE and Hygiene BIOLOGIC SAFETY General Considerations Spills Bloodborne Pathogens Airborne Pathogens Shipping CHEMICAL SAFETY Hazard Communication Safety Data Sheet OSHA Laboratory Standard Toxic Effects from Hazardous Substances Storage and Handling of Chemicals RADIATION SAFETY Environmental Protection Personal Protection Nonionizing Radiation FIRE SAFETY The Chemistry of Fire Classification of Fires Types and Applications of Fire Extinguishers CONTROL OF OTHER HAZARDS Electrical Hazards Compressed Gas Hazards  Cryogenic Materials Hazards Mechanical Hazards Ergonomic Hazards DISPOSAL OF HAZARDOUS MATERIALS Chemical Waste Radioactive Waste Biohazardous Waste ACCIDENT DOCUMENTATION AND INVESTIGATION QUESTIONS BIBLIOGRAPHY AND SUGGESTED READING 3 Method Evaluation and Quality Control Michael W. Rogers, Cindi Bullock Letsos, Matthew P. A. Henderson, Monte S. Willis, and Christoper R. MCCudden BASIC CONCEPTS Descriptive Statistics: Measures of Center, Spread, and Shape Descriptive Statistics of Groups of Paired Observations Inferential Statistics METHOD EVALUATION Regulatory Aspects of Method Evaluation (Alphabet Soup) Method Selection Method Evaluation First Things First: Determine Imprecision and Inaccuracy Measurement of Imprecision Interference Studies COM Studies Allowable Analytical Error Method Evaluation Acceptance Criteria QUALITY CONTROL QC Charts Operation of a QC System Multirules RULE! Proficiency Testing REFERENCE INTERVAL STUDIES Establishing Reference Intervals Selection of Reference Interval Study Individuals Preanalytic and Analytic Considerations Determining Whether to Establish or Transfer and Verify Reference Intervals Analysis of Reference Values Data Analysis to Establish a Reference Interval Data Analysis to Transfer and Verify a Reference Interval DIAGNOSTIC EFFICIENCY Measures of Diagnostic Efficiency SUMMARY PRACTICE PROBLEMS Problem 3-1. Calculation of Sensitivity and Specificity Problem 3-2. A Quality Control Decision Problem 3-3. Precision (Replication) Problem 3-4. Recovery Problem 3-5. Interference Problem 3-6. Sample Labeling Problem 3-7. QC Program for POCT Testing Problem 3-8. QC Rule Interpretation Problem 3-9. Reference Interval Study Design  QUESTIONS ONLINE RESOURCES REFERENCES 4 Lean Six Sigma Methodology Basics and Quality Improvement in the Clinical Chemistry Laboratory Cindi Bullock Letsos, Michael W. Rogers, Christoper R. McCudden, and Monte S. Willis LEAN SIX SIGMA METHODOLOGY ADOPTION AND IMPLEMENTATION OF LEAN SIX SIGMA PROCESS IMPROVEMENT MEASUREMENTS OF SUCCESS USING LEAN AND SIX SIGMA LEAN SIX SIGMA APPLICATIONS IN THE LABORATORY AND THE GREATER HEALTH CARE SYSTEM PRACTICAL APPLICATION OF SIX SIGMA METRICS Detecting Laboratory Errors Defining the Sigma Performance of an Assay Choosing the Appropriate Westgard Rules PRESENT AND EVOLVING APPROACHES TO QUALITY IMPROVEMENT AND QUALITY ASSURANCE IN THE CLINICAL LABORATORY: FROM QCP TO IQCP AND ISO5189 Today's Standard: Quality Control Plan QUALITY CONTROL PLAN BASED ON RISK MANAGEMENT: CLSI EP23-A DOCUMENT QUALITY ASSESSMENT INDIVIDUAL QUALITY CONTROL PROGRAM: AN OPTION TO STREAMLINE QCP ISO5189—QUALITY MANAGEMENT IN MEDICAL  LABORATORIES: ADDING RISK ASSESSMENT TO THE FORMULA INTERNATIONALLY (GLOBAL INITIATIVE) CONCLUSIONS ACKNOWLEDGMENTS QUESTIONS REFERENCES RESOURCES FOR FURTHER INFORMATION ON 5 Analytic Techniques Julia C. Drees, Matthew S. Petrie, and Alan H. B. Wu SPECTROPHOTOMETRY Beer Law Spectrophotometric Instruments Components of a Spectrophotometer Spectrophotometer Quality Assurance Atomic Absorption Spectrophotometer Flame Photometry Fluorometry Basic Instrumentation Chemiluminescence Turbidity and Nephelometry Laser Applications ELECTROCHEMISTRY Galvanic and Electrolytic Cells Half-Cells Ion-Selective Electrodes pH Electrodes Gas-Sensing Electrodes  Enzyme Electrodes Coulometric Chloridometers and Anodic Stripping Voltammetry ELECTROPHORESIS Procedure Support Materials Treatment and Application of Sample Detection and Quantitation Electroendosmosis Isoelectric Focusing Capillary Electrophoresis Two-Dimensional Electrophoresis OSMOMETRY Freezing Point Osmometer SURFACE PLASMON RESONANCE QUESTIONS REFERENCES 6 Chromatography and Mass Spectrometry Julia C. Drees, Matthew S. Petrie, and Alan H. B. Wu CHROMATOGRAPHY Modes of Separation Chromatographic Procedures High-Performance Liquid Chromatography Gas Chromatography MASS SPECTROMETRY Sample Introduction and Ionization Mass Analyzer Detector  APPLICATIONS OF MS IN THE CLINICAL LABORATORY Small Molecule Analysis Mass Spectrometry in Proteomics and Pathogen Identification Mass Spectrometry at the Point of Care QUESTIONS REFERENCES 7 Principles of Clinical Chemistry Automation Ryan W. Greer and Joely A. Straseski HISTORY OF AUTOMATED ANALYZERS DRIVING FORCES TOWARD MORE AUTOMATION BASIC APPROACHES TO AUTOMATION STEPS IN AUTOMATED ANALYSIS Specimen Preparation and Identification Specimen Measurement and Delivery Reagent Systems and Delivery Chemical Reaction Phase Measurement Phase Signal Processing and Data Handling SELECTION OF AUTOMATED ANALYZERS TOTAL LABORATORY AUTOMATION Preanalytic Phase (Sample Processing) Analytic Phase (Chemical Analyses) Postanalytic Phase (Data Management) FUTURE TRENDS IN AUTOMATION QUESTIONS REFERENCES 8 Immunochemical Techniques  Alan H. B. Wu IMMUNOASSAYS General Considerations Unlabeled Immunoassays Labeled Immunoassays Future Directions for Immunoassays QUESTIONS REFERENCES 9 Molecular Theory and Techniques Shashi Mehta NUCLEIC ACID–BASED TECHNIQUES Nucleic Acid Chemistry Nucleic Acid Extraction Hybridization Techniques DNA Sequencing DNA Chip Technology Target Amplification Probe Amplification Signal Amplification Nucleic Acid Probe Applications QUESTIONS REFERENCES 10 Point-of-Care Testing Heather Crookston LABORATORY REGULATIONS Accreditation  POCT Complexity IMPLEMENTATION Establishing Need POCT Implementation Protocol Personnel Requirements QUALITY MANAGEMENT Accuracy Requirements QC and Proficiency Testing POC APPLICATIONS INFORMATICS AND POCT QUESTIONS REFERENCES PART two Clinical Correlations and Analytic Procedures 11 Amino Acids and Proteins Takara L. Blamires AMINO ACIDS Overview Basic Structure Metabolism Essential Amino Acids Nonessential Amino Acids Recently Identified Amino Acids Aminoacidopathies Methods of Analysis PROTEINS Overview Basic Structure General Chemical Properties Synthesis Catabolism and Nitrogen Balance Classification PLASMA PROTEINS Prealbumin Albumin Globulins OTHER PROTEINS OF CLINICAL SIGNIFICANCE Myoglobin Cardiac Troponin Brain Natriuretic Peptide and N-Terminal–Brain Natriuretic Peptide Fibronectin Adiponectin β-Trace Protein Cross-Linked C-Telopeptides Cystatin C Amyloid TOTAL PROTEIN ABNORMALITIES Hypoproteinemia Hyperproteinemia METHODS OF ANALYSIS Total Nitrogen  Total Protein Fractionation, Identification, and Quantitation of Specific Proteins Serum Protein Electrophoresis High-Resolution Protein Electrophoresis Capillary Electrophoresis Isoelectric Focusing Immunochemical Methods PROTEINS IN OTHER BODY FLUIDS Urinary Protein CSF Protein QUESTIONS REFERENCES 12 Nonprotein Nitrogen Compounds Elizabeth L. Frank UREA Biochemistry Clinical Application Analytical Methods Pathophysiology URIC ACID Biochemistry Clinical Application Analytical Methods Pathophysiology CREATININE/CREATINE Biochemistry  Clinical Application Analytical Methods Pathophysiology AMMONIA Biochemistry Clinical Application Analytical Methods Pathophysiology QUESTIONS REFERENCES 13 Enzymes Kamisha L. Johnson-Davis GENERAL PROPERTIES AND DEFINITIONS ENZYME CLASSIFICATION AND NOMENCLATURE ENZYME KINETICS Catalytic Mechanism of Enzymes Factors That Influence Enzymatic Reactions Measurement of Enzyme Activity Calculation of Enzyme Activity Measurement of Enzyme Mass Enzymes as Reagents ENZYMES OF CLINICAL SIGNIFICANCE Creatine Kinase Lactate Dehydrogenase Aspartate Aminotransferase Alanine Aminotransferase Alkaline Phosphatase  Acid Phosphatase γ-Glutamyltransferase Amylase Lipase Glucose-6-Phosphate Dehydrogenase Drug-Metabolizing Enzymes QUESTIONS REFERENCES 14 Carbohydrates Vicki S. Freeman GENERAL DESCRIPTION OF CARBOHYDRATES Classification of Carbohydrates Stereoisomers Monosaccharides, Disaccharides, and Polysaccharides Chemical Properties of Carbohydrates Glucose Metabolism Fate of Glucose Regulation of Carbohydrate Metabolism HYPERGLYCEMIA Diabetes Mellitus Pathophysiology of Diabetes Mellitus Criteria for Testing for Prediabetes and Diabetes Criteria for the Diagnosis of Diabetes Mellitus Criteria for the Testing and Diagnosis of GDM HYPOGLYCEMIA Genetic Defects in Carbohydrate Metabolism ROLE OF LABORATORY IN DIFFERENTIAL DIAGNOSIS  AND MANAGEMENT OF PATIENTS WITH GLUCOSE METABOLIC ALTERATIONS Methods of Glucose Measurement Self-Monitoring of Blood Glucose Glucose Tolerance and 2-Hour Postprandial Tests Glycosylated Hemoglobin/HbA1c Ketones Albuminuria Islet Autoantibody and Insulin Testing QUESTIONS REFERENCES 15 Lipids and Lipoproteins Raffick A. R. Bowen, Amar A. Sethi, G. Russell Warnick, and Alan T. Remaley LIPID CHEMISTRY Fatty Acids Triglycerides Phospholipids Cholesterol GENERAL LIPOPROTEIN STRUCTURE Chylomicrons Very-Low-Density Lipoproteins Intermediate-Density Lipoproteins Low-Density Lipoproteins Lipoprotein (a) High-Density Lipoproteins Lipoprotein X LIPOPROTEIN PHYSIOLOGY AND METABOLISM Lipid Absorption Exogenous Pathway Endogenous Pathway Reverse Cholesterol Transport Pathway LIPID AND LIPOPROTEIN POPULATION DISTRIBUTIONS Dyslipidemia and Children National Cholesterol Education Program National Heart, Lung, and Blood Institute DIAGNOSIS AND TREATMENT OF LIPID DISORDERS Arteriosclerosis Hyperlipoproteinemia Hypercholesterolemia PCSK9 Hypertriglyceridemia Combined Hyperlipidemia Lp(a) Elevation Non–HDL Cholesterol Hypobetalipoproteinemia Hypoalphalipoproteinemia LIPID AND LIPOPROTEIN ANALYSES Lipid Measurement Cholesterol Measurement Triglyceride Measurement Lipoprotein Methods HDL Methods LDL Methods  Compact Analyzers Apolipoprotein Methods Phospholipid Measurement Fatty Acid Measurement STANDARDIZATION OF LIPID AND LIPOPROTEIN ASSAYS Precision Accuracy Matrix Interactions CDC Cholesterol Reference Method Laboratory Network Analytic Performance Goals Quality Control Specimen Collection QUESTIONS REFERENCES 16 Electrolytes James March Mistler WATER Osmolality THE ELECTROLYTES Sodium Potassium Chloride Bicarbonate Magnesium Calcium Phosphate Lactate  ANION GAP ELECTROLYTES AND RENAL FUNCTION QUESTIONS REFERENCES 17 Blood Gases, pH, and Buffer Systems Yachana Kataria and Mark D. Kellogg ACID–BASE BALANCE Maintenance of H+ Buffer Systems: Regulation of H+ and the Henderson- Hasselbalch Equation Regulation of Acid–Base Balance: Lungs and Kidneys (Transport of Carbon Dioxide) ASSESSMENT OF ACID–BASE HOMEOSTASIS The Bicarbonate Buffering System Acid–Base Disorders: Acidosis and Alkalosis OXYGEN AND GAS EXCHANGE Oxygen and Carbon Dioxide Oxygen Transport Assessment of a Patient's Oxygen Status Hemoglobin–Oxygen Dissociation MEASUREMENT Spectrophotometric Determination of Oxygen Saturation (CO- Oximetry) Blood Gas Analyzers: pH, pCO2, and pO2 Measurement of pO2 Measurement of pH and pCO2 Types of Electrochemical Sensors  Optical Sensors Calibration Correction for Temperature Calculated Parameters QUALITY ASSURANCE Preanalytic Considerations Analytic Assessments: Quality Control and Proficiency Testing QUESTIONS REFERENCES 18 Trace and Toxic Elements Frederick G. Strathmann and Carrie J. Haglock-Adler OVERVIEW AND OBJECTIVES INSTRUMENTATION AND METHODS Sample Collection and Processing Atomic Emission Spectroscopy Atomic Absorption Spectroscopy Inductively Coupled Plasma Mass Spectrometry Interferences Elemental Speciation Alternative Analytical Techniques ALUMINUM Introduction Absorption, Transport, and Excretion Health Effects and Toxicity Laboratory Evaluation of Aluminum Status ARSENIC Introduction  Health Effects and Toxicity Absorption, Transport, and Excretion Laboratory Evaluation of Arsenic Status CADMIUM Introduction Absorption, Transport, and Excretion Health Effects and Toxicity Laboratory Evaluation of Cadmium Status CHROMIUM Introduction Absorption, Transport, and Excretion Health Effects, Deficiency, and Toxicity Laboratory Evaluation of Chromium Status COPPER Introduction Absorption, Transport, and Excretion Health Effects, Deficiency, and Toxicity Laboratory Evaluation of Copper Status IRON Introduction Absorption, Transport, and Excretion Health Effects, Deficiency, and Toxicity Laboratory Evaluation of Iron Status LEAD Introduction Absorption, Transport, and Excretion Health Effects and Toxicity  Laboratory Evaluation of Lead Status MERCURY Introduction Absorption, Transport, and Excretion Health Effects and Toxicity Laboratory Evaluation of Mercury Status MANGANESE Introduction Absorption, Transport, and Excretion Health Effects, Deficiency, and Toxicity Laboratory Evaluation of Manganese Status MOLYBDENUM Introduction Absorption, Transport, and Excretion Health Effects, Deficiency, and Toxicity Laboratory Evaluation of Molybdenum Status SELENIUM Introduction Absorption, Transport, and Excretion Health Effects, Deficiency, and Toxicity Laboratory Evaluation of Selenium Status ZINC Introduction Absorption, Transport, and Excretion Health Effects, Deficiency, and Toxicity Laboratory Evaluation of Zinc Status QUESTIONS  BIBLIOGRAPHY REFERENCES 19 Porphyrins and Hemoglobin Elizabeth L. Frank and Sara A. Taylor PORPHYRINS Porphyrin Properties Biochemistry: Synthesis of Heme Pathophysiology: Disorders of Heme Biosynthesis Clinical Application Analytical Methods HEMOGLOBIN Role in the Body Structure of Hemoglobin Synthesis and Degradation of Hemoglobin Clinical Significance and Disease Correlation Analytical Methods DNA Technology MYOGLOBIN Structure and Role in the Body Clinical Significance Analytical Methods QUESTIONS REFERENCES PART three Assessment of Organ System Functions 20 Hypothalamic and Pituitary Function Robert E. Jones and Heather Corn EMBRYOLOGY AND ANATOMY FUNCTIONAL ASPECTS OF THE HYPOTHALAMIC– HYPOPHYSEAL UNIT HYPOPHYSIOTROPIC OR HYPOTHALAMIC HORMONES ANTERIOR PITUITARY HORMONES PITUITARY TUMORS GROWTH HORMONE Actions of GH Testing Acromegaly GH Deficiency PROLACTIN Prolactinoma Other Causes of Hyperprolactinemia Clinical Evaluation of Hyperprolactinemia Management of Prolactinoma Idiopathic Galactorrhea HYPOPITUITARISM Etiology of Hypopituitarism Treatment of Panhypopituitarism POSTERIOR PITUITARY HORMONES Oxytocin Vasopressin QUESTIONS REFERENCES 21 Adrenal Function Vishnu Sundaresh and Deepika S. Reddy THE ADRENAL GLAND: AN OVERVIEW EMBRYOLOGY AND ANATOMY THE ADRENAL CORTEX BY ZONE Cortex Steroidogenesis Congenital Adrenal Hyperplasia PRIMARY ALDOSTERONISM Overview Etiology Diagnosis Treatment Isolated Hypoaldosteronism ADRENAL CORTICAL PHYSIOLOGY ADRENAL INSUFFICIENCY Overview Symptoms Diagnosis Treatment HYPERCORTISOLISM (CUSHING'S SYNDROME) Overview Etiology Diagnosis Treatment ADRENAL ANDROGENS Androgen Excess Diagnosis  Treatment THE ADRENAL MEDULLA Embryology Biosynthesis, Storage, and Secretion of Catecholamines Metabolism and Excretion of Catecholamines PHEOCHROMOCYTOMA AND PARAGANGLIOMA Overview Epidemiology Clinical Presentation Diagnosis Interfering medications Biochemical Testing Plasma-Free Metanephrines 24-Hour Urine Fractionated Metanephrines and Catecholamines Normal Results Case Detection 24-Hour Urine Fractionated Metanephrines and Catecholamines Plasma-Fractionated Metanephrines Radiographic Localization Treatment Outcome, Prognosis, and Follow-up Genetic Testing ADRENAL INCIDENTALOMA CASE STUDIES QUESTIONS REFERENCES 22 Gonadal Function  Mahima Gulati THE TESTES Functional Anatomy of the Male Reproductive Tract Physiology of the Testicles Disorders of Sexual Development and Testicular Hypofunction Diagnosis of Hypogonadism Testosterone Replacement Therapy Monitoring Testosterone Replacement Therapy THE OVARIES Early Ovarian Development Functional Anatomy of the Ovaries Hormonal Production by the Ovaries The Menstrual Cycle Hormonal Control of Ovulation Pubertal Development in the Female Precocious Sexual Development Menstrual Cycle Abnormalities Hirsutism Estrogen Replacement Therapy QUESTIONS REFERENCES 23 The Thyroid Gland Marissa Grotzke THE THYROID Thyroid Anatomy and Development Thyroid Hormone Synthesis  Protein Binding of Thyroid Hormone Control of Thyroid Function Actions of Thyroid Hormone TESTS FOR THYROID FUNCTION Blood Tests OTHER TOOLS FOR THYROID EVALUATION Nuclear Medicine Evaluation Thyroid Ultrasound Fine-Needle Aspiration DISORDERS OF THE THYROID Hypothyroidism Thyrotoxicosis Graves' Disease Toxic Adenoma and Multinodular Goiter DRUG-INDUCED THYROID DYSFUNCTION Amiodarone-Induced Thyroid Disease Subacute Thyroiditis NONTHYROIDAL ILLNESS THYROID NODULES QUESTIONS REFERENCES 24 Calcium Homeostasis and Hormonal Regulation Josephine Abraham and Dev Abraham CALCIUM HOMEOSTASIS HORMONAL REGULATION OF CALCIUM METABOLISM Vitamin D Parathyroid Hormone  ORGAN SYSTEM REGULATION OF CALCIUM METABOLISM GI Regulation Role of Kidneys Bone Physiology HYPERCALCEMIA Causes of Hypercalcemia Primary Hyperparathyroidism Familial Hypocalciuric Hypercalcemia Hyperthyroidism Addison's Disease Milk Alkali Syndrome Medications That Cause Hypercalcemia HYPOCALCEMIA Causes of Hypocalcemia METABOLIC BONE DISEASES Rickets and Osteomalacia Osteoporosis SECONDARY HYPERPARATHYROIDISM IN RENAL FAILURE QUESTIONS REFERENCES 25 Liver Function Janelle M. Chiasera and Xin Xu ANATOMY Gross Anatomy Microscopic Anatomy  BIOCHEMICAL FUNCTIONS Excretory and Secretory Metabolism Detoxification and Drug Metabolism LIVER FUNCTION ALTERATIONS DURING DISEASE Jaundice Cirrhosis Tumors Reye's Syndrome Drug- and Alcohol-Related Disorders ASSESSMENT OF LIVER FUNCTION/LIVER FUNCTION TESTS Bilirubin METHODS Urobilinogen in Urine and Feces Serum Bile Acids Enzymes Tests Measuring Hepatic Synthetic Ability Tests Measuring Nitrogen Metabolism Hepatitis QUESTIONS REFERENCES 26 Laboratory Markers of Cardiac Damage and Function Ronald R. Henriquez, Michael Durando, Brian C. Jensen, Christoper R. McCudden, and Monte S. Willis CARDIAC ISCHEMIA, ANGINA, AND HEART ATTACKS THE PATHOPHYSIOLOGY OF ATHEROSCLEROSIS, THE  DISEASE PROCESS UNDERLYING MI MARKERS OF CARDIAC DAMAGE Initial Markers of Cardiac Damage Cardiac Troponins CK-MB and Troponin I/Troponin T Considerations in Kidney Disease Patients Other Markers of Cardiac Damage CARDIAC INJURY OCCURS IN MANY DISEASE PROCESSES, BEYOND MI THE LABORATORY WORKUP OF PATIENTS SUSPECTED OF HEART FAILURE AND THE USE OF CARDIAC BIOMARKERS IN HEART FAILURE THE USE OF NATRIURETIC PEPTIDES AND TROPONINS IN THE DIAGNOSIS AND RISK STRATIFICATION OF HEART FAILURE Cardiac Troponins MARKERS OF CHD RISK C-Reactive Protein Homocysteine MARKERS OF PULMONARY EMBOLISM Use of D-Dimer Detection in PE Value of Assaying Troponin and BNP in Acute PE SUMMARY QUESTIONS REFERENCES 27 Renal Function Kara L. Lynch and Alan H. B. Wu RENAL ANATOMY  RENAL PHYSIOLOGY Glomerular Filtration Tubular Function Elimination of Nonprotein Nitrogen Compounds Water, Electrolyte, and Acid–Base Homeostasis Endocrine Function ANALYTIC PROCEDURES Creatinine Clearance Estimated GFR Cystatin C β2-Microglobulin Myoglobin Albuminuria Neutrophil Gelatinase–Associated Lipocalin NephroCheck Urinalysis PATHOPHYSIOLOGY Glomerular Diseases Tubular Diseases Urinary Tract Infection/Obstruction Renal Calculi Renal Failure QUESTIONS REFERENCES 28 Pancreatic Function and Gastrointestinal Function Edward P. Fody PHYSIOLOGY OF PANCREATIC FUNCTION  DISEASES OF THE PANCREAS TESTS OF PANCREATIC FUNCTION Secretin/CCK Test Fecal Fat Analysis Sweat Electrolyte Determinations Serum Enzymes FECAL ELASTASE PHYSIOLOGY AND BIOCHEMISTRY OF GASTRIC SECRETION CLINICAL ASPECTS OF GASTRIC ANALYSIS TESTS OF GASTRIC FUNCTION Measuring Gastric Acid in Basal and Maximal Secretory Tests Measuring Gastric Acid Plasma Gastrin INTESTINAL PHYSIOLOGY CLINICOPATHOLOGIC ASPECTS OF INTESTINAL FUNCTION TESTS OF INTESTINAL FUNCTION Lactose Tolerance Test D-Xylose Absorption Test D-Xylose Test Serum Carotenoids Other Tests of Intestinal Malabsorption QUESTIONS SUGGESTED READING REFERENCES 29 Body Fluid Analysis Kyle B. Riding  CEREBROSPINAL FLUID SEROUS FLUIDS Pleural Fluid Pericardial Fluid Peritoneal Fluid AMNIOTIC FLUID Hemolytic Disease of the Newborn Neural Tube Defects Fetal Lung Maturity Phosphatidylglycerol Lamellar Body Counts SWEAT SYNOVIAL FLUID QUESTIONS REFERENCES PART four Specialty Areas of Clinical Chemistry 30 Therapeutic Drug Monitoring Takara L. Blamires OVERVIEW ROUTES OF ADMINISTRATION DRUG ABSORPTION DRUG DISTRIBUTION FREE VERSUS BOUND DRUGS DRUG METABOLISM DRUG ELIMINATION PHARMACOKINETICS SPECIMEN COLLECTION PHARMACOGENOMICS CARDIOACTIVE DRUGS Digoxin Quinidine Procainamide Disopyramide ANTIBIOTICS Aminoglycosides Teicoplanin Vancomycin ANTIEPILEPTIC DRUGS Phenobarbital and Primidone Phenytoin and Fosphenytoin Valproic Acid Carbamazepine Ethosuximide Felbamate Gabapentin Lamotrigine Levetiracetam Oxcarbazepine Tiagabine Topiramate Zonisamide  PSYCHOACTIVE DRUGS Lithium Tricyclic Antidepressants Clozapine Olanzapine IMMUNOSUPPRESSIVE DRUGS Cyclosporine Tacrolimus Sirolimus Mycophenolic Acid ANTINEOPLASTICS Methotrexate BRONCHODILATORS Theophylline QUESTIONS SUGGESTED READINGS REFERENCES 31 Toxicology Takara L. Blamires XENOBIOTICS, POISONS, AND TOXINS ROUTES OF EXPOSURE DOSE–RESPONSE RELATIONSHIP Acute and Chronic Toxicity ANALYSIS OF TOXIC AGENTS TOXICOLOGY OF SPECIFIC AGENTS Alcohols Carbon Monoxide  Caustic Agents Cyanide Metals and Metalloids Pesticides TOXICOLOGY OF THERAPEUTIC DRUGS Salicylates Acetaminophen TOXICOLOGY OF DRUGS OF ABUSE Amphetamines Anabolic Steroids Cannabinoids Cocaine Opiates Phencyclidine Sedatives–Hypnotics QUESTIONS REFERENCES 32 Circulating Tumor Markers: Basic Concepts and Clinical Applications Christoper R. McCudden and Monte S. Willis TYPES OF TUMOR MARKERS APPLICATIONS OF TUMOR MARKER DETECTION Screening and Susceptibility Testing Prognosis Monitoring Effectiveness of Therapy and Disease Recurrence LABORATORY CONSIDERATIONS FOR TUMOR MARKER MEASUREMENT  Immunoassays High-Performance Liquid Chromatography Immunohistochemistry and Immunofluorescence Enzyme Assays FREQUENTLY ORDERED TUMOR MARKERS α-Fetoprotein METHODOLOGY Cancer Antigen 125 Carcinoembryonic Antigen Human Chorionic Gonadotropin Prostate-Specific Antigen FUTURE DIRECTIONS QUESTIONS SUGGESTED READING REFERENCES 33 Nutrition Assessment Linda S. Gorman and Maria G. Boosalis NUTRITION CARE PROCESS: OVERVIEW NUTRITION ASSESSMENT BIOCHEMICAL MARKERS: MACRONUTRIENTS Protein Fat Carbohydrate BIOCHEMICAL MARKERS: MISCELLANEOUS Parenteral Nutrition Electrolytes Urine Testing  Organ Function BIOCHEMICAL MARKERS: MICRONUTRIENTS Vitamins Conditionally Essential Nutrients Minerals Trace Elements QUESTIONS REFERENCES 34 Clinical Chemistry and the Geriatric Patient Laura M. Hickes and J. Marvin McBride THE AGING OF AMERICA AGING AND MEDICAL SCIENCE GENERAL PHYSIOLOGIC CHANGES WITH AGING Muscle Bone Gastrointestinal System Kidney/Urinary System Immune System Endocrine System Sex Hormones Glucose Metabolism EFFECTS OF AGE ON LABORATORY TESTING Muscle Bone Gastrointestinal System Urinary System Immune System  Endocrine System Sex Hormones Glucose Metabolism ESTABLISHING REFERENCE INTERVALS FOR THE ELDERLY PREANALYTICAL VARIABLES UNIQUE TO GERIATRIC PATIENTS DISEASES PREVALENT IN THE ELDERLY AGE-ASSOCIATED CHANGES IN DRUG METABOLISM Absorption Distribution Metabolism Elimination ATYPICAL PRESENTATIONS OF COMMON DISEASES Geriatric Syndromes THE IMPACT OF EXERCISE AND NUTRITION ON CHEMISTRY RESULTS IN THE ELDERLY QUESTIONS REFERENCES 35 Clinical Chemistry and the Pediatric Patient Tracey G. Polsky and Michael J. Bennett DEVELOPMENTAL CHANGES FROM NEONATE TO ADULT Respiration and Circulation Growth Organ Development Problems of Prematurity and Immaturity PHLEBOTOMY AND CHOICE OF INSTRUMENTATION FOR PEDIATRIC SAMPLES Phlebotomy Preanalytic Concerns Choice of Analyzer POINT-OF-CARE ANALYSIS IN PEDIATRICS REGULATION OF BLOOD GASES AND PH IN NEONATES AND INFANTS Blood Gas and Acid–Base Measurement REGULATION OF ELECTROLYTES AND WATER: RENAL FUNCTION Disorders Affecting Electrolytes and Water Balance DEVELOPMENT OF LIVER FUNCTION Physiologic Jaundice Energy Metabolism Diabetes Nitrogen Metabolism Nitrogenous End Products as Markers of Renal Function Liver Function Tests CALCIUM AND BONE METABOLISM IN PEDIATRICS Hypocalcemia and Hypercalcemia ENDOCRINE FUNCTION IN PEDIATRICS Hormone Secretion Hypothalamic–Pituitary–Thyroid System Hypothalamic–Pituitary–Adrenal Cortex System Growth Factors Endocrine Control of Sexual Maturation DEVELOPMENT OF THE IMMUNE SYSTEM Basic Concepts of Immunity  Components of the Immune System Neonatal and Infant Antibody Production Immunity Disorders GENETIC DISEASES Cystic Fibrosis Newborn Screening for Whole Populations Diagnosis of Metabolic Disease in the Clinical Setting DRUG METABOLISM AND PHARMACOKINETICS Therapeutic Drug Monitoring Toxicologic Issues in Pediatric Clinical Chemistry QUESTIONS REFERENCES Index  PART one Basic Principles and Practice of Clinical Chemistry  1 Basic Principles and Practices KATHRYN DUGAN and ELIZABETH WARNING Chapter Outline Units of Measure Reagents Chemicals Reference Materials Water Specifications Solution Properties Concentration Colligative Properties Redox Potential Conductivity pH and Buffers Clinical Laboratory Supplies Thermometers/Temperature Glassware and Plasticware Desiccators and Desiccants Balances Centrifugation Laboratory Mathematics and Calculations Significant Figures Logarithms Concentration Dilutions Water of Hydration Graphing and Beer's Law Specimen Considerations Types of Samples Sample Processing Sample Variables Chain of Custody Electronic and Paper Reporting of Results Questions References Chapter Objectives Upon completion of this chapter, the clinical laboratorian should be able to do the following: Convert results from one unit format to another using the SI and traditional systems. Describe the classifications used for reagent grade water. Identify the varying chemical grades used in reagent preparation and indicate their correct use. Define primary standard and standard reference materials. Describe the following terms that are associated with solutions and, when appropriate, provide the respective units: percent, molarity, normality, molality, saturation, colligative properties, redox potential, and conductivity. Define a buffer and give the formula for pH and pK calculations. Use the Henderson-Hasselbalch equation to determine the missing variable when given either the pK and pH or the pK and concentration of the weak acid and its conjugate base. List and describe the types of thermometers used in the clinical laboratory. Classify the type of pipette when given an actual pipette or its description. Demonstrate the proper use of a measuring and volumetric pipette. Describe two ways to calibrate a pipetting device. Define a desiccant and discuss how it is used in the clinical laboratory. Describe how to properly care for and balance a centrifuge. Correctly perform the laboratory mathematical calculations provided in this chapter. Identify and describe the types of samples used in clinical chemistry. Outline the general steps for processing blood samples. Apply Beer's law to determine the concentration of a sample when the absorbance or change in absorbance is provided. Identify the preanalytic variables that can adversely affect laboratory results as presented in this chapter. Key Terms Analyte Anhydrous Arterial blood Beer's law Buffer Centrifugation Cerebrospinal fluid (CSF) Colligative property Conductivity Deionized water Deliquescent substance Delta absorbance Density Desiccant Desiccator Dilution Dilution factor Distilled water Equivalent weight Erlenmeyer flasks Filtration Graduated cylinder Griffin Beaker Hemolysis Henderson-Hasselbalch equation Hydrate Hygroscopic Icterus International unit Ionic strength Lipemia Molality Molarity Normality One-point calibration Osmotic pressure Oxidized Oxidizing agent Percent solution pH Pipette Primary standard Ratio Reagent grade water Redox potential Reduced Reducing agent Reverse osmosis Serial dilution Serum Significant figures Solute Solution Solvent Specific gravity Standard Standard reference materials (SRMs) Système International d'Unités (SI) Thermistor  Ultrafiltration Valence Whole blood The primary purpose of a clinical chemistry laboratory is to perform analytic procedures that yield accurate and precise information, aiding in patient diagnosis and treatment. The achievement of reliable results requires that the clinical laboratory scientist be able to correctly use basic supplies and equipment and possess an understanding of fundamental concepts critical to any analytic procedure. The topics in this chapter include units of measure, basic laboratory supplies, and introductory laboratory mathematics, plus a brief discussion of specimen collection, processing, and reporting. UNITS OF MEASURE Any meaningful quantitative laboratory result consists of two components: the first component represents the number related to the actual test value and the second is a label identifying the units. The unit defines the physical quantity or dimension, such as mass, length, time, or volume.1 Not all laboratory tests have well-defined units, but whenever possible, the units used should be reported. Although several systems of units have traditionally been utilized by various scientific divisions, the Système International d'Unités (SI), adopted internationally in 1960, is preferred in scientific literature and clinical laboratories and is the only system employed in many countries. This system was devised to provide the global scientific community with a uniform method of describing physical quantities. The SI system units (referred to as SI units) are based on the metric system. Several subclassifications exist within the SI system, one of which is the basic unit. There are seven basic units (Table 1.1), with length (meter), mass (kilogram), and quantity of a substance (mole) being the units most frequently encountered. Another set of SI-recognized units is termed derived units. A derived unit, as the name implies, is a derivative or a mathematical function describing one of the basic units. An example of an SI- derived unit is meters per second (m/s), used to express velocity. Some non-SI units are so widely used that they have become acceptable for use within the SI system (Table 1.1). These include long-standing units such as hour, minute, day, gram, liter, and plane angles expressed as degrees. The SI uses standard prefixes that, when added to a given basic unit, can indicate decimal fractions or multiples of that unit (Table 1.2). For example, 0.001 liter can be expressed using the prefix milli, or 10−3, and since it requires moving the decimal point three places to the right, it can then be written as 1 milliliter, or abbreviated as 1 mL. It may also be written in scientific notation as 1 × 10−3 L. Likewise, 1,000 liters would use the prefix of kilo (103) and could be written as 1 kiloliter or expressed in scientific notation as 1 × 103 L. TABLE 1.1 SI Units TABLE 1.2 Prefixes Used with SI Units Prefixes are used to indicate a subunit or multiple of a basic SI unit. It is important to understand the relationship these prefixes have to the basic unit. The highlighted upper portion of Table 1.2 indicates prefixes that are smaller than the basic unit and are frequently used in clinical laboratories. When converting between prefixes, simply note the relationship between the two prefixes based on whether you are changing to a smaller or larger prefix. For example, if converting from one liter (1.0 × 100 or 1.0) to milliliters (1.0 × 10−3 or 0.001), the starting unit (L) is larger than the desired unit by a factor of 1,000 or 103. This means that the decimal place would be moved to the right three places, so 1.0 liter (L) equals 1,000 milliliters (mL). When changing 1,000 milliliters (mL) to 1.0 liter (L), the process is reversed and the decimal point would be moved three places to the left to become 1.0 L. Note that the SI term for mass is kilogram; it is the only basic unit that contains a prefix as part of its name. Generally, the standard prefixes for mass use the term gram rather than kilogram. Example 1: Convert 1.0 L to μL 1.0 L (1 × 100) = ? μL (micro = 10−6); move the decimal place six places to the right and it becomes 1,000,000 μL; reverse the process to determine the expression in L (move the decimal six places to the left of 1,000,000 μL to get 1.0 L). Example 2: Convert 5 mL to μL 5 mL (milli = 10−3, larger) = ? μL (micro = 10−6, smaller); move the decimal by three places to the right and it becomes 5,000 μL. Example 3: Convert 5.3 mL to dL 5.3 mL (milli = 10−3, smaller) = ? dL (deci = 10−1, larger); move the  decimal place by two places to the left and it becomes 0.053 dL. Reporting of laboratory results is often expressed in terms of substance concentration (e.g., moles) or the mass of a substance (e.g., mg/dL, g/dL, g/L, mmol/L, and IU) rather than in SI units. These familiar and traditional units can cause confusion during interpretation. Appendix D (on thePoint), Conversion of Traditional Units to SI Units for Common Clinical Chemistry Analytes, lists both reference and SI units together with the conversion factor from traditional to SI units for common analytes. As with other areas of industry, the laboratory and the rest of medicine are moving toward adopting universal standards promoted by the International Organization for Standardization, often referred to as ISO. This group develops standards of practice, definitions, and guidelines that can be adopted by everyone in a given field, providing for more uniform terminology and less confusion. Many national initiatives have recommended common units for laboratory test results, but none have been widely adopted.2 As with any transition, clinical laboratory scientists should be familiar with all the terms currently used in their field. REAGENTS In today's highly automated laboratory, there seems to be little need for reagent preparation by the clinical laboratory scientist. Most instrument manufacturers make the reagents in a ready-to-use form or “kit” where all necessary reagents and respective storage containers are prepackaged as a unit requiring only the addition of water or buffer to the prepackaged components for reconstitution. A heightened awareness of the hazards of certain chemicals and the numerous regulatory agency requirements has caused clinical chemistry laboratories to readily eliminate massive stocks of chemicals and opt instead for the ease of using prepared reagents. Periodically, especially in hospital laboratories involved in research and development, biotechnology applications, specialized analyses, or method validation, the laboratorian may still face preparing various reagents or solutions. Chemicals Analytic chemicals exist in varying grades of purity: analytic reagent (AR); ultrapure, chemically pure (CP); United States Pharmacopeia (USP); National Formulary (NF); and technical or commercial grade.3 A committee of the American Chemical Society (ACS) established specifications for AR grade chemicals, and chemical manufacturers will either meet or exceed these requirements. Labels on reagents state the actual impurities for each chemical lot or list the maximum allowable impurities. The labels should be clearly printed with the percentage of impurities present and either the initials AR or ACS or the term For laboratory use or ACS Standard-Grade Reference Materials. Chemicals of this category are suitable for use in most analytic laboratory procedures. Ultrapure chemicals have been put through additional purification steps for use in specific procedures such as chromatography, atomic absorption, immunoassays, molecular diagnostics, standardization, or other techniques that require extremely pure chemicals. These reagents may carry designations of HPLC (high-performance liquid chromatography) or chromatographic on their labels. Because USP and NF grade chemicals are used to manufacture drugs, the limitations established for this group of chemicals are based only on the criterion of not being injurious to individuals. Chemicals in this group may be pure enough for use in most chemical procedures; however, it should be recognized that the purity standards are not based on the needs of the laboratory and, therefore, may or may not meet all assay requirements. Reagent designations of CP or pure grade indicate that the impurity limitations are not stated and that preparation of these chemicals is not uniform. It is not recommended that clinical laboratories use these chemicals for reagent preparation unless further purification or a reagent blank is included. Technical or commercial grade reagents are used primarily in manufacturing and should never be used in the clinical laboratory. Organic reagents also have varying grades of purity that differ from those used to classify inorganic reagents. These grades include a practical grade with some impurities; CP, which approaches the purity level of reagent grade chemicals; spectroscopic (spectrally pure) and chromatographic grade organic reagents, with purity levels attained by their respective procedures; and reagent grade (ACS), which is certified to contain impurities below certain levels established by the ACS. As in any analytic method, the desired organic reagent purity is dictated by the particular application. Other than the purity aspects of the chemicals, laws related to the Occupational Safety and Health Administration (OSHA)4 require manufacturers to indicate any physical or biologic health hazards and precautions needed for the safe use, storage, and disposal of any chemical. A manufacturer is required to provide technical data sheets for each chemical manufactured on a document called a Safety Data Sheet (SDS). Reference Materials Unlike other areas of chemistry, clinical chemistry is involved in the analysis of biochemical by-products found in biological fluids, such as serum, plasma, or urine, making purification and a known exact composition of the material almost impossible. For this reason, traditionally defined standards used in analytical chemistry do not readily apply in clinical chemistry. A primary standard is a highly purified chemical that can be measured directly to produce a substance of exact known concentration and purity. The ACS has purity tolerances for primary standards, because most biologic constituents are unavailable within these tolerance limitations; the National Institute of Standards and Technology (NIST)-certified standard reference materials (SRMs) are used instead of ACS primary standard materials.5, 6, 7 The NIST developed certified reference materials/SRMs for use in clinical chemistry laboratories. They are assigned a value after careful analysis, using state-of-the-art methods and equipment. The chemical composition of these substances is then certified; however, they may not possess the purity equivalent of a primary standard. Because each substance has been characterized for certain chemical or physical properties, it can be used in place of an ACS primary standard in clinical work and is often used to verify calibration or accuracy/bias assessments. Many manufacturers use an NIST SRM when producing calibrator and standard materials, and in this way, these materials are considered “traceable to NIST” and may meet certain accreditation requirements. There are SRMs for a number of routine analytes, hormones, drugs, and blood gases, with others being added.5 Water Specifications8 Water is the most frequently used reagent in the laboratory. Because tap water is unsuitable for laboratory applications, most procedures, including reagent and standard preparation, use water that has been substantially purified. There are various methods for water purification including distillation, ion exchange, reverse osmosis, ultrafiltration, ultraviolet light, sterilization, and ozone treatment. Laboratory requirements generally call for reagent grade water that, according to the Clinical and Laboratory Standards Institute (CLSI), is classified into one of six categories based on the specifications needed for its use rather than the method of purification or preparation.9,10 These categories include clinical laboratory reagent water (CLRW), special reagent water (SRW), instrument feed water, water supplied by method manufacturer, autoclave and wash water, and commercially bottled purified water. Laboratories need to assess whether the water meets the specifications needed for its application. Most water-monitoring parameters include at least microbiological count, pH, resistivity (measure of resistance in ohms and influenced by the number of ions present), silicate, particulate matter, and organics. Each category has a specific acceptable limit. A long-held convention for categorizing water purity was based on three types, I through III, with type I water having the most stringent requirements and generally suitable for routine laboratory use. Prefiltration can remove particulate matter from municipal water supplies before any additional treatments. Filtration cartridges are composed of glass; cotton; activated charcoal, which removes organic materials and chlorine; and submicron filters (≤0.2 mm), which remove any substances larger than the filter's pores, including bacteria. The use of these filters depends on the quality of the municipal water and the other purification methods used. For example, hard water (containing calcium, iron, and other dissolved elements) may require prefiltration with a glass or cotton filter rather than activated charcoal or submicron filters, which quickly become clogged and are expensive to use. The submicron filter may be better suited after distillation, deionization, or reverse osmosis treatment. Distilled water has been purified to remove almost all organic materials, using a technique of distillation much like that found in organic chemistry laboratory distillation experiments in which water is boiled and vaporized. Many impurities do not rise in the water vapor and will remain in the boiling apparatus so that the water collected after condensation has less contamination. Water may be distilled more than once, with each distillation cycle removing additional impurities. Ultrafiltration and nanofiltration, like distillation, are excellent in removing particulate matter, microorganisms, and any pyrogens or endotoxins. Deionized water has some or all ions removed, although organic material may still be present, so it is neither pure nor sterile. Generally, deionized water is purified from previously treated water, such as prefiltered or distilled water. Deionized water is produced using either an anion or a cation exchange resin, followed by replacement of the removed ions with hydroxyl or hydrogen ions. The ions that are anticipated to be removed from the water will dictate the type of ion exchange resin to be used. One column cannot service all ions present in water. A combination of several resins will produce different grades of deionized water. A two-bed system uses an anion resin followed by a cation resin. The different resins may be in separate columns or in the same column. This process is excellent in removing dissolved ionized solids and dissolved gases. Reverse osmosis is a process that uses pressure to force water through a semipermeable membrane, producing water that reflects a filtered product of the original water. It does not remove dissolved gases. Reverse osmosis may be used for the pretreatment of water. Ultraviolet oxidation, which removes some trace organic material or sterilization processes at specific wavelengths, when used in combination with ozone treatment, can destroy bacteria but may leave behind residual products. These techniques are often used after other purification processes have been completed. Production of reagent grade water largely depends on the condition of the feed water. Generally, reagent grade water can be obtained by initially filtering it to remove particulate matter, followed by reverse osmosis, deionization, and a 0.2-mm filter or more restrictive filtration process. Type III/autoclave wash water is acceptable for glassware washing but not for analysis or reagent preparation. Traditionally, type II water was acceptable for most analytic requirements, including reagent, quality control, and standard preparation, while type I water was used for test methods requiring minimum interference, such as trace metal, iron, and enzyme analyses. Use with HPLC may require less than a 0.2-mm final filtration step and falls into the SRW category. Some molecular diagnostic or mass spectrophotometric techniques may require special reagent grade water; some reagent grade water should be used immediately, so storage is discouraged because the resistivity changes. Depending on the application, CLRW should be stored in a manner that reduces any chemical or bacterial contamination and for short periods. Testing procedures to determine the quality of reagent grade water include measurements of resistance, pH, colony counts on selective and nonselective media for the detection of bacterial contamination, chlorine, ammonia, nitrate or nitrite, iron, hardness, phosphate, sodium, silica, carbon dioxide, chemical oxygen demand, and metal detection. Some accreditation agencies11 recommend that laboratories document culture growth, pH, and specific resistance on water used in reagent preparation. Resistance is measured because pure water, devoid of ions, is a poor conductor of electricity and has increased resistance. The relationship of water purity to resistance is linear. Generally, as purity increases, so does resistance. This one measurement does not suffice for determination of true water purity because a nonionic contaminant may be present that has little effect on resistance. Note that reagent water meeting specifications from other organizations, such as the ASTM, may not be equivalent to those established by the CLSI, and care should be taken to meet the assay procedural requirements for water type requirements. Solution Properties In clinical chemistry, substances found in biologic fluids including serum, plasma, urine, and spinal fluid are quantified. A substance that is dissolved in a liquid is called a solute; in laboratory science, these biologic solutes are also known as analytes. The liquid in which the solute is dissolved—in this instance, a biologic fluid—is the solvent. Together they represent a solution. Any chemical or biologic solution is described by its basic properties, including concentration, saturation, colligative properties, redox potential, conductivity, density, pH, and ionic strength. Concentration Analyte concentration in solution can be expressed in many ways. Routinely, concentration is expressed as percent solution, molarity, molality, or normality, Note that these are non-SI units, and the SI expression for the amount of a substance is the mole. Percent solution is expressed as the amount of solute per 100 total units of solution. Three expressions of percent solutions are weight per weight (w/w), volume per volume (v/v), and weight per volume (w/v). Weight per weight (% w/w) refers to the number of grams of solute per 100 g of solution. Volume per volume (% v/v) is used for liquid solutes and gives the milliliters of solute in 100 mL of solution. For v/v solutions, it is recommended that grams per deciliter (g/dL) be used instead of % v/v. Weight per volume (% w/v) is the most commonly used percent solution in the clinical laboratory and is defined as the number of grams of solute in 100 mL of solution. This is not the same as molarity and care must be taken to not confuse the two. Molarity (M) is expressed as the number of moles per 1 L of solution. One mole of a substance equals its gram molecular weight (gmw), so the customary units of molarity (M) are moles/liter. The SI representation for the traditional molar concentration is moles of solute per volume of solution, with the volume of the solution given in liters. The SI expression for concentration should be represented as moles per liter (mol/L), millimoles per liter (mmol/L), micromoles per liter (μmol/L), and nanomoles per liter (nmol/L). The familiar concentration term molarity has not been adopted by the SI as an expression of concentration. It should also be noted that molarity depends on volume, and any significant physical changes that influence volume, such as changes in temperature and pressure, will also influence molarity. Molality (m) represents the amount of solute per 1 kg of solvent. Molality is sometimes confused with molarity; however, it can be easily distinguished from molarity because molality is always expressed in terms of moles per kilogram (weight per weight) and describes moles per 1,000 g (1 kg) of solvent. Note that the common abbreviation (m) for molality is a lowercase “m,” while the uppercase (M) refers to molarity. The preferred expression for molality is moles per kilogram (mol/kg) to avoid any confusion. Unlike molarity, molality is not influenced by temperature or pressure because it is based on mass rather than volume. Normality is the least likely of the four concentration expressions to be encountered in clinical laboratories, but it is often used in chemical titrations and chemical reagent classification. It is defined as the number of gram equivalent weights per 1 L of solution. An equivalent weight is equal to the gmw of a substance divided by its valence. The valence is the number of units that can combine with or replace 1 mole of hydrogen ions for acids and hydroxyl ions for bases and the number of electrons exchanged in oxidation–reduction reactions. Normality is always equal to or greater than the molarity of the compound. Normality was previously used for reporting electrolyte values, such as sodium [Na+], potassium [K+], and chloride [Cl−], expressed as milliequivalents per liter (mEq/L); however, this convention has been replaced with the more familiar units of millimoles per liter (mmol/L). Solution saturation gives little specific information about the concentration of solutes in a solution. A solution is considered saturated when no more solvent can be dissolved in the solution. Temperature, as well as the presence of other ions, can influence the solubility constant for a solute in a given solution and thus affect the saturation. Routine terms in the clinical laboratory that describe the extent of saturation are dilute, concentrated, saturated, and supersaturated. A dilute solution is one in which there is relatively little solute or one that has a lower solute concentration per volume of solvent than the original, such as when making a dilution. In contrast, a concentrated solution has a large quantity of solute in solution. A solution in which there is an excess of undissolved solute particles can be referred to as a saturated solution. As the name implies, a supersaturated solution has an even greater concentration of undissolved solute particles than a saturated solution of the same substance. Because of the greater concentration of solute particles, a supersaturated solution is thermodynamically unstable. The addition of a crystal of solute or mechanical agitation disturbs the supersaturated solution, resulting in crystallization of any excess material out of solution. An example is seen when measuring serum osmolality by freezing point depression. Colligative Properties Colligative properties are those properties related to the number of solute particles per solvent molecules, not on the type of particles present. The behavior of particles or solutes in solution demonstrates four repeatable properties, osmotic pressure, vapor pressure, freezing point, and boiling point, these are called colligative properties. Vapor pressure is the pressure exerted by the vapor when the liquid solvent is in equilibrium with the vapor. Freezing point is the temperature at which the first crystal (solid) of solvent forms in equilibrium with the solution. Boiling point is the temperature at which the vapor pressure of the solvent reaches atmospheric pressure (usually one atmosphere). Osmotic pressure is the pressure that opposes osmosis when a solvent flows through a semipermeable membrane to establish equilibrium between compartments of differing concentration. The osmotic pressure of a dilute solution is directly proportional to the concentration of the molecules in solution. The expression for concentration is the osmole. One osmole of a substance equals the molarity or molality multiplied by the number of particles, not the kind of particle, at dissociation. If molarity is used, the resulting expression would be termed osmolarity; if molality is used, the expression changes to osmolality. Osmolality is preferred since it depends on the weight rather than volume and is not readily influenced by temperature and pressure changes. When a solute is dissolved in a solvent, the colligative properties change in a predictable manner for each osmole of substance present. In the clinical setting, freezing point and vapor pressure depression can be measured as a function of osmolality. Freezing point is preferred since vapor pressure measurements can give inaccurate readings when some substances, such as alcohols, are present in the samples. Redox Potential Redox potential, or oxidation–reduction potential, is a measure of the ability of a solution to accept or donate electrons. Substances that donate electrons are called reducing agents; those that accept electrons are considered oxidizing agents. The pneumonic—LEO (lose electrons oxidized) the lion says GER (gain electrons reduced)—may prove useful when trying to recall the relationship between reducing/oxidizing agents and redox potential. Conductivity Conductivity is a measure of how well electricity passes through a solution. A solution's conductivity quality depends principally on the number of respective charges of the ions present. Resistivity, the reciprocal of conductivity, is a measure of a substance's resistance to the passage of electrical current. The primary application of resistivity in the clinical laboratory is for assessing the purity of water. Resistivity or resistance is expressed as ohms and conductivity is expressed as ohms−1. pH and Buffers Buffers are weak acids or bases and their related salts that, as a result of their dissociation characteristics, minimize changes in the hydrogen ion concentration. Hydrogen ion concentration is often expressed as pH. A lowercase p in front of certain letters or abbreviations operationally means the “negative logarithm of” or “inverse log of” that substance. In keeping with this convention, the term pH represents the negative or inverse log of the hydrogen ion concentration. Mathematically, pH is expressed as (Eq. 1-1) where [H+] equals the concentration of hydrogen ions in moles per liter (M). The pH scale ranges from 0 to 14 and is a convenient way to express hydrogen ion concentration. Unlike a strong acid or base, which dissociates almost completely, the dissociation constant for a weak acid or base solution (like a buffer) tends to be very small, meaning little dissociation occurs. The dissociation of acetic acid (CH3COOH), a weak acid, can be illustrated as follows:  (Eq. 1-2) HA = weak acid, A− = conjugate base, H+ = hydrogen ions, [] = concentration of anything in the bracket. Sometimes, the conjugate base (A−) will be referred to as a “salt” since, physiologically, it will be associated with some type of cation such as sodium (Na+). Note that the dissociation constant, Ka, for a weak acid may be calculated using the following equation: (Eq. 1-3) Rearrangement of this equation reveals (Eq. 1-4) Taking the log of each quantity and then multiplying by minus 1 (−1), the equation can be rewritten as (Eq. 1-5) By convention, lowercase p means “negative log of”; therefore, −log[H+] may be written as pH, and −Ka may be written as pKa. The equation now becomes (Eq. 1-6) Eliminating the minus sign in front of the log of the quantity results in an equation known as the Henderson-Hasselbalch equation, which mathematically describes the dissociation characteristics of weak acids (pKa) and bases (pKb) and the effect on pH: (Eq. 1-7) When the ratio of [A−] to [HA] is 1, the pH equals the pK and the buffer has its greatest buffering capacity. The dissociation constant Ka, and therefore the pKa, remains the same for a given substance. Any changes in pH are solely due to the ratio of conjugate base [A−] concentration to weak acid [HA] concentration. Ionic strength is another important aspect of buffers, particularly in separation techniques. Ionic strength is the concentration or activity of ions in a solution or buffer. Increasing ionic strength increases the ionic cloud surrounding a compound and decreases the rate of particle migration. It can also promote compound dissociation into ions effectively increasing the solubility of some salts, along with changes in current, which can also affect electrophoretic separation. CLINICAL LABORATORY SUPPLIES In today's clinical chemistry laboratory, many different types of equipment are in use. Most of the manual techniques have been replaced by automation, but it is still necessary for the laboratory scientist to be knowledgeable in the operation and use of certain equipment. The following is a brief discussion of the composition and general use of common equipment found in a clinical chemistry laboratory, including thermometers, pipettes, flasks, beakers, and dessicators. Thermometers/Temperature The predominant practice for temperature measurement uses the Celsius (°C) scale; however, Fahrenheit (°F) and Kelvin (°K) scales are also used.12 The SI designation for temperature is the Kelvin scale. Table 1.3 gives the conversion formulas between Fahrenheit and Celsius scales and Appendix C (thePoint) lists the various conversion formulas. TABLE 1.3 Common Temperature Conversions  All analytic reactions occur at an optimal temperature. Some laboratory procedures, such as enzyme determinations, require precise temperature control, whereas others work well over a wide range of temperatures. Reactions that are temperature dependent use some type of heating/cooling cell, heating/cooling block, or water/ice bath to provide the correct temperature environment. Laboratory refrigerator temperatures are often critical and need periodic verification. Thermometers either are an integral part of an instrument or need to be placed in the device for temperature maintenance. The two types of thermometers discussed include liquid-in-glass and electronic thermometer or thermistor probe; however, several other types of temperature-indicating devices are in use. Regardless of w