Teaching Monograph
Fundamental Issues in Clinical Bernard E. Statland, MD, PhD Department of Pathology University of California, Davis Sacramento Medical Center Sacramento, California
Copyright ( 1979, University Associated for Research and Development in All rights reserved The American Journal of Pathology Official publication of The American Association of Pathologists
Published by The American Association of Pathologists, 9650 Rockville Pike, Bethesda, Maryland
Pathology, Inc.
FUNDAMENTAL ISSUES IN CLINICAL CHEMISTRY Preanalyc Sources of Variation Unrelated to Patlogy Prior Exercise
Presious Food Intake Prior Ethanol Consumption Drug Ingestion Posture Other Factors
Evaluating the Performance of an Analyic Method Precision Accuracv Dynamic Range Freedom From Interferences Assessing the Practicability of an Analy tic Method
Physioloc Variation as a Function of Time Sources of Intra-indisidual N ariation
Within-Dav- Xariation Dav-to-Dav Variation
Reference Values in Clinical Chemistry Establishing Reference Values Criticisms of the Consentional \Method of Establishing Reference \Values Age Sex Race Pregnancy
Subject-Based Reference Inten-als
Mulivariate Analysis of Clinical and Laboratory Data Classification Using Multisariate Analy sis Algorithms in Clinical Chemistry Probabilistic Mlodel for Discrete Variates Probabilistic Model for Continuous Variates Linear Discriminant Anal sis
Condusion
244(2) 245(3: 246(4) 247(5) 248(6) 248(6) 249(7)
50(8) 250(8)
2551(9) 2.52(10) 2.52(10)
2.3)11)
253111)
2.53 11)
254)1) 2.5(13)
5816) 2.58(16) 2.59(17)
259(17) 260(18) 260(18) 260(18) 260( 18)
261(19) 262(20) 262(20) 265)(2.3)
267(2.5) 268(26) 268(26 )
Foreword to Teaching Monographs This teaching monograph is being published by The American Journal of Pathology for Universities Associated for Research and Education in Pathology as a service to medical students and their teachers of pathology. This venture represents a joint effort to make such teaching material available to a wide audience. Separately bound copies of this Teaching Monograph can be purchased from Universities Associated for Research and Education in Pathology, Inc., 9650 Rockville Pike, Bethesda, MD 20014. The charge is $2.25 per copy for orders of up to ten and $1.25 per copy for orders of ten or more (prepaid).
The Editorial Board John R. Carter, MD, Case Western Reserve University School of Medicine
Francis E. Cuppage, MD, University of Kansas Medical Center Joe W. Grisham, MD, The University of North Carolina School of Medicine Robert B. Jennings, MD, Duke University Medical School Werner H. Kirsten, MD, The University of Chicago Vincent R. Marchesi, MD, Yale University School of Medicine Goetz W. Richter, MD, The University of Rochester School of Medicine Dante G. Scarpelli, MD, Northwestern University Medical School Robert E. Stowell, MD, University of California, Davis Benjamin F. Trump, MD, University of Maryland Series Editor: Dante G. Scarpelli, MD
Fundamental Issues in Clinical Chemistry Bernard E. Statland, MD, PhD
CLINICAL CHEMISTRY iS that discipline which is involved in the measurement of chemical constituents in biologic material, the interpretation of these results in terms of improving the quality of health care, and the pursuit of a biochemical understanding underlying the pathogenesis of disease, the maintenance of health, and the responsiveness to various therapeutic maneuvers. In the United States, the hospital laboratories are managed by clinical pathologists. The hospital laboratories are divided into the following main areas: blood bank, laboratory hematology, laboratory immunology, microbiology, and clinical chemistry. In that the measurements performed in clinical chemistry encompass such a wide variety of specimens and analytes, up to 50% of the tests in a hospital laboratory are performed within the clinical chemistry department. The objective of the hospital-based clinical chemistry laboratory is to provide the clinician with timely, accurate, and relevant chemical measurements on specimens obtained from patients. These measurements are used by the clinicians in evaluating their patients. The measurements are performed using simple to complex instruments and laboratory-made reagents or reagents in a kit form. Numerous reagent companies produce kits for most assays performed in the clinical chemistry laboratory. The clinical chemistry laboratory has a varied personnel responsible for many steps in the analytic procedure. The director of the clinical chemistry laboratory is responsible for interacting with the various clinicians in the medical center, giving a clinical relevance to the ordering and establishing of chemical measurements in the laboratory, and managing the day-to-day operation of the laboratory. The individual responsible for the quality control in the clinical chemistry laboratory evaluates the performance of the various assays in terms of the precision and accuracy of such assays. The technologists in the laboratory are primarily involved with and responsible for the quality of the results produced in the laboratory. The technologist must be able to perform basic laboratory maneuvers and must be competent to work with complex instrumentation used to analyze various constituents in biologic specimens. Before discussing certain fundamental issues which are in the mainstream of clinical chemistry, I would like to highlight certain of the major trends in clinical chemistry over the past 2 decades. These trends have included development of new instrumentation capable of performing chemical assays more precisely and faster and on smaller volumes of
0002-9440/79/0411-0239$01.00 O UAREP
243 (1)
244
STATLAND
(2)
American Journal of Pathology
biologic material; an ability to assay constituents in very low concentrations; an increased tendency for clinicians to rely on clinical chemistry data for clinical decision-making; a resultant dramatic increase in the number (both type and amount) of chemical measurements performed in laboratories; and a questioning by many sectors in our society of the possible overutilization of laboratory services. The easy accessibility of an ever increasing array of chemical assays coupled with the high costs of these assays have demanded that we begin to concentrate some of our efforts in maximizing the use of the data we are producing as well as in identifying the nonpathologic factors which contribute to the variability of the chemistry results. Bearing this in mind, I have selected to review certain fundamental issues of clinical chemistry which relate to those concerns, ie, a) preanalytic sources of variation, b) evaluating the performance of an analytic method, c) physiologic variation as a function of time, d) reference values in clinical chemistry, and e) multivariate analysis of clinical and laboratory data. Preanalytic Sources of Variation Unrelated to Pathology The analytic procedure is fundamental to clinical chemistry. The major
steps of the procedure are presented in Table 1. It begins with the collection of a specimen obtained from a patient; it involves the transport, Table 1-Steps Involved in Requesting, Performing, and Evaluating a Measured Quantity
I. The physician requests a measurement of a quantity. Laboratory personnel perform the assay. A. Pre-instrumental phase 1. Preparation of the patient 2. Obtaining the specimen 3. Processing the specimen 4. Storing the specimen prior to the measuring step B. Instrumental phase 1. Dispensing the sample into a reaction vessel 2. Combining the sample with one or more reagents 3. Recording some physical-chemical consequences of the reaction 4. Calculating the value of the quantity measured C. Post-instrumental phase 1. Laboratory and technical staff accept the value (result of the measurement) as being of good quality. 2. The report of the measurement is sent to the requesting physician. Ill. The physician evaluates the resultant measurement. 1. The physician assesses whether the measurement could be consistent with other known patient information. 2. The physician makes a clinical decision partially based on the reported measureII.
ment.
Adapted from Statland BE, Winkel P: Sources of variation in laboratory measurements. Clinical Diagnosis by Laboratory Methods. Edited by JB Henry, et al. Philadelphia, W. B. Saunders, 1979, p 4
Vol. 95, No. 1 April 1979
CLINICAL CHEMISTRY
245
(3)
storage, and pretreatment of the biologic material (eg, blood, urine, cerebrospinal fluid); and it concludes with the measurement step(s) in producing a laboratory result. The producer (industry) and the user (practicing clinical chemist) have worked in concert over the past years to identify various sources of analytic variations and to eliminate, or at least to minimize, these sources. In the main, this collaborative effort has been successful. Local, regional, and national programs of quality control and proficiency testing have directed clinical chemists to particular assays which were "out of control" and to instruments that were not performing up to expected standards. The improvement in analytic precision has permitted the clinical chemist to examine noninstrumental sources of variation with a higher degree of confidence. It soon became apparent that for many assays in the chemistry laboratory it was the preinstrumental phase which demanded more attention. The preanalytic sources of variation are the factors involved in the preparation of the patient, which are unrelated to the pathologic entity for which the patient is examined. Prior Exercise
Numerous reports suggest that physical exercise causes a profound increase in the activity values of enzymes commonly measured in human serum, notably creatine kinase (CPK). King, Statland, and Savory 1 examined the effect of 1 hour of strenuous physical activity (a vigorous game of handball) on the activity values of four enzymes in serums of healthy young men. The percent change for serum creatine kinase, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (AP), and total protein as monitored before and various intervals after the exercise challenge in one of the volunteers is presented in Figure 1. The results depicted in Figure 1 were typical for the group ie, the mean peak increase for CPK was +125%, for AST was +41%, and for LDH was +37%. The enzymes in muscle rose while AP (not present in high concentrations in muscle) did not show substantial changes. Those factors which tend to bring about the most dramatic increases in serum CPK values include the use of untrained volunteers, the administration of a strenuous exercise challenge, and waiting 8 to 20 hours after the challenge before obtaining blood specimens. Because an elevated serum CPK value is often relied on to diagnose certain pathologic entities (eg, myocardial infarction, myopathic disorders), the clinician must inquire into the history of prior strenuous physical activity. In addition, the availability of the assay for the CPK-MB isoenzyme (an evaluation of CPK-MB is a very sensitive indicator of cardiac damage) will aid in the differential diagnosis
American Journal of Pathology
STATLAND
246 (4)
Figure 1-Serum
en-
zyme activity values for
w :
g W
J w
Fundamental Issues in Clinical Bernard E. Statland, MD, PhD Department of Pathology University of California, Davis Sacramento Medical Center Sacramento, California
Copyright ( 1979, University Associated for Research and Development in All rights reserved The American Journal of Pathology Official publication of The American Association of Pathologists
Published by The American Association of Pathologists, 9650 Rockville Pike, Bethesda, Maryland
Pathology, Inc.
FUNDAMENTAL ISSUES IN CLINICAL CHEMISTRY Preanalyc Sources of Variation Unrelated to Patlogy Prior Exercise
Presious Food Intake Prior Ethanol Consumption Drug Ingestion Posture Other Factors
Evaluating the Performance of an Analyic Method Precision Accuracv Dynamic Range Freedom From Interferences Assessing the Practicability of an Analy tic Method
Physioloc Variation as a Function of Time Sources of Intra-indisidual N ariation
Within-Dav- Xariation Dav-to-Dav Variation
Reference Values in Clinical Chemistry Establishing Reference Values Criticisms of the Consentional \Method of Establishing Reference \Values Age Sex Race Pregnancy
Subject-Based Reference Inten-als
Mulivariate Analysis of Clinical and Laboratory Data Classification Using Multisariate Analy sis Algorithms in Clinical Chemistry Probabilistic Mlodel for Discrete Variates Probabilistic Model for Continuous Variates Linear Discriminant Anal sis
Condusion
244(2) 245(3: 246(4) 247(5) 248(6) 248(6) 249(7)
50(8) 250(8)
2551(9) 2.52(10) 2.52(10)
2.3)11)
253111)
2.53 11)
254)1) 2.5(13)
5816) 2.58(16) 2.59(17)
259(17) 260(18) 260(18) 260(18) 260( 18)
261(19) 262(20) 262(20) 265)(2.3)
267(2.5) 268(26) 268(26 )
Foreword to Teaching Monographs This teaching monograph is being published by The American Journal of Pathology for Universities Associated for Research and Education in Pathology as a service to medical students and their teachers of pathology. This venture represents a joint effort to make such teaching material available to a wide audience. Separately bound copies of this Teaching Monograph can be purchased from Universities Associated for Research and Education in Pathology, Inc., 9650 Rockville Pike, Bethesda, MD 20014. The charge is $2.25 per copy for orders of up to ten and $1.25 per copy for orders of ten or more (prepaid).
The Editorial Board John R. Carter, MD, Case Western Reserve University School of Medicine
Francis E. Cuppage, MD, University of Kansas Medical Center Joe W. Grisham, MD, The University of North Carolina School of Medicine Robert B. Jennings, MD, Duke University Medical School Werner H. Kirsten, MD, The University of Chicago Vincent R. Marchesi, MD, Yale University School of Medicine Goetz W. Richter, MD, The University of Rochester School of Medicine Dante G. Scarpelli, MD, Northwestern University Medical School Robert E. Stowell, MD, University of California, Davis Benjamin F. Trump, MD, University of Maryland Series Editor: Dante G. Scarpelli, MD
Fundamental Issues in Clinical Chemistry Bernard E. Statland, MD, PhD
CLINICAL CHEMISTRY iS that discipline which is involved in the measurement of chemical constituents in biologic material, the interpretation of these results in terms of improving the quality of health care, and the pursuit of a biochemical understanding underlying the pathogenesis of disease, the maintenance of health, and the responsiveness to various therapeutic maneuvers. In the United States, the hospital laboratories are managed by clinical pathologists. The hospital laboratories are divided into the following main areas: blood bank, laboratory hematology, laboratory immunology, microbiology, and clinical chemistry. In that the measurements performed in clinical chemistry encompass such a wide variety of specimens and analytes, up to 50% of the tests in a hospital laboratory are performed within the clinical chemistry department. The objective of the hospital-based clinical chemistry laboratory is to provide the clinician with timely, accurate, and relevant chemical measurements on specimens obtained from patients. These measurements are used by the clinicians in evaluating their patients. The measurements are performed using simple to complex instruments and laboratory-made reagents or reagents in a kit form. Numerous reagent companies produce kits for most assays performed in the clinical chemistry laboratory. The clinical chemistry laboratory has a varied personnel responsible for many steps in the analytic procedure. The director of the clinical chemistry laboratory is responsible for interacting with the various clinicians in the medical center, giving a clinical relevance to the ordering and establishing of chemical measurements in the laboratory, and managing the day-to-day operation of the laboratory. The individual responsible for the quality control in the clinical chemistry laboratory evaluates the performance of the various assays in terms of the precision and accuracy of such assays. The technologists in the laboratory are primarily involved with and responsible for the quality of the results produced in the laboratory. The technologist must be able to perform basic laboratory maneuvers and must be competent to work with complex instrumentation used to analyze various constituents in biologic specimens. Before discussing certain fundamental issues which are in the mainstream of clinical chemistry, I would like to highlight certain of the major trends in clinical chemistry over the past 2 decades. These trends have included development of new instrumentation capable of performing chemical assays more precisely and faster and on smaller volumes of
0002-9440/79/0411-0239$01.00 O UAREP
243 (1)
244
STATLAND
(2)
American Journal of Pathology
biologic material; an ability to assay constituents in very low concentrations; an increased tendency for clinicians to rely on clinical chemistry data for clinical decision-making; a resultant dramatic increase in the number (both type and amount) of chemical measurements performed in laboratories; and a questioning by many sectors in our society of the possible overutilization of laboratory services. The easy accessibility of an ever increasing array of chemical assays coupled with the high costs of these assays have demanded that we begin to concentrate some of our efforts in maximizing the use of the data we are producing as well as in identifying the nonpathologic factors which contribute to the variability of the chemistry results. Bearing this in mind, I have selected to review certain fundamental issues of clinical chemistry which relate to those concerns, ie, a) preanalytic sources of variation, b) evaluating the performance of an analytic method, c) physiologic variation as a function of time, d) reference values in clinical chemistry, and e) multivariate analysis of clinical and laboratory data. Preanalytic Sources of Variation Unrelated to Pathology The analytic procedure is fundamental to clinical chemistry. The major
steps of the procedure are presented in Table 1. It begins with the collection of a specimen obtained from a patient; it involves the transport, Table 1-Steps Involved in Requesting, Performing, and Evaluating a Measured Quantity
I. The physician requests a measurement of a quantity. Laboratory personnel perform the assay. A. Pre-instrumental phase 1. Preparation of the patient 2. Obtaining the specimen 3. Processing the specimen 4. Storing the specimen prior to the measuring step B. Instrumental phase 1. Dispensing the sample into a reaction vessel 2. Combining the sample with one or more reagents 3. Recording some physical-chemical consequences of the reaction 4. Calculating the value of the quantity measured C. Post-instrumental phase 1. Laboratory and technical staff accept the value (result of the measurement) as being of good quality. 2. The report of the measurement is sent to the requesting physician. Ill. The physician evaluates the resultant measurement. 1. The physician assesses whether the measurement could be consistent with other known patient information. 2. The physician makes a clinical decision partially based on the reported measureII.
ment.
Adapted from Statland BE, Winkel P: Sources of variation in laboratory measurements. Clinical Diagnosis by Laboratory Methods. Edited by JB Henry, et al. Philadelphia, W. B. Saunders, 1979, p 4
Vol. 95, No. 1 April 1979
CLINICAL CHEMISTRY
245
(3)
storage, and pretreatment of the biologic material (eg, blood, urine, cerebrospinal fluid); and it concludes with the measurement step(s) in producing a laboratory result. The producer (industry) and the user (practicing clinical chemist) have worked in concert over the past years to identify various sources of analytic variations and to eliminate, or at least to minimize, these sources. In the main, this collaborative effort has been successful. Local, regional, and national programs of quality control and proficiency testing have directed clinical chemists to particular assays which were "out of control" and to instruments that were not performing up to expected standards. The improvement in analytic precision has permitted the clinical chemist to examine noninstrumental sources of variation with a higher degree of confidence. It soon became apparent that for many assays in the chemistry laboratory it was the preinstrumental phase which demanded more attention. The preanalytic sources of variation are the factors involved in the preparation of the patient, which are unrelated to the pathologic entity for which the patient is examined. Prior Exercise
Numerous reports suggest that physical exercise causes a profound increase in the activity values of enzymes commonly measured in human serum, notably creatine kinase (CPK). King, Statland, and Savory 1 examined the effect of 1 hour of strenuous physical activity (a vigorous game of handball) on the activity values of four enzymes in serums of healthy young men. The percent change for serum creatine kinase, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (AP), and total protein as monitored before and various intervals after the exercise challenge in one of the volunteers is presented in Figure 1. The results depicted in Figure 1 were typical for the group ie, the mean peak increase for CPK was +125%, for AST was +41%, and for LDH was +37%. The enzymes in muscle rose while AP (not present in high concentrations in muscle) did not show substantial changes. Those factors which tend to bring about the most dramatic increases in serum CPK values include the use of untrained volunteers, the administration of a strenuous exercise challenge, and waiting 8 to 20 hours after the challenge before obtaining blood specimens. Because an elevated serum CPK value is often relied on to diagnose certain pathologic entities (eg, myocardial infarction, myopathic disorders), the clinician must inquire into the history of prior strenuous physical activity. In addition, the availability of the assay for the CPK-MB isoenzyme (an evaluation of CPK-MB is a very sensitive indicator of cardiac damage) will aid in the differential diagnosis
American Journal of Pathology
STATLAND
246 (4)
Figure 1-Serum
en-
zyme activity values for
w :
g W
J w
