Vol. 20 No. 17 Vol. 15 No. 1
Standardization of Sodium and Potassium Ion-Selective Electrode Systems to the Flame Photometric Reference Method; Approved Standard—Second Edition
This document contains recommendations on the expression of the results of ion-selective electrode measurement of sodium and potassium ion activities in undiluted serum, plasma, or whole blood in clinical practice.
A standard for global application developed through the NCCLS consensus process.
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Standardization of Sodium and Potassium Ion-Selective Electrode Systems to the Flame Photometric Reference Method; Approved Standard—Second Edition
Abstract
Standardization of Sodium and Potassium Ion-Selective Electrode Systems to the Flame Photometric Reference Method; Approved StandardCSecond Edition (NCCLS document C29-A2) offers a protocol for standardizing instruments that contain direct ion-selective electrodes to give results in concentration terms that are verifiable to the reference method (flame photometry) for specimens with normal plasma water (i.e., lipids and proteins). The document describes the preparation of serum pools to carry out the procedure. Laboratories without the resources, equipment, or personnel to prepare the pools can purchase them from the National Institute of Standards and Technology (Gaithersburg, MD). It is recommended in C29-A2 that the accuracy of each new direct potentiometric instrument be verified with these standard pools, together with the flame photometer, if it is also used to report patient results.
NCCLS. Standardization of Sodium and Potassium Ion-Selective Electrode Systems to the Flame Photometric Reference Method; Approved Standard—Second Edition. NCCLS document C29-A2 (ISBN 1-56238-410-4). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA 2000.
THE NCCLS consensus process, which is the mechanism for moving a document through two or more levels of review by the healthcare community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of NCCLS documents. Current editions are listed in the NCCLS Catalog, which is distributed to member organizations, and to nonmembers on request. If your organization is not a member and would like to become one, and to request a copy of the NCCLS Catalog, contact the NCCLS Executive Offices. Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail: [email protected];
Website: www.nccls.org
ii
ISBN 1-56238-410-4 ISSN 0273-3099
Standardization of Sodium and Potassium Ion-Selective Electrode Systems to the Flame Photometric Reference Method; Approved Standard—Second Edition
Volume 20 Number 17
Paul D’Orazio, Ph.D
W. Gregory Miller, Ph.D., Chairholder Gary L. Myers, Ph.D., Vice-Chairholder Basil T. Doumas, Ph.D.
John H. Eckfeldt, M.D., Ph.D.
Susan A. Evans, Ph.D.
Gary A. Graham, Ph.D., DABCC Patrick J. Parsons, Ph.D.
Noel V. Stanton, M.S.
iv
This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system, transmitted, or made available in any form or by any means (electronic, mechanical, photocopying, recording, or otherwise) without prior written permission of NCCLS, except as stated below.
NCCLS hereby grants permission to reproduce limited portions of this publication for use in laboratory procedure manuals at a single site, for interlibrary loan, or for use in educational programs provided that multiple copies of such reproduction shall include the following notice, be distributed without charge, and, in no event, contain more than 20% of the document’s text.
Reproduced with permission, from NCCLS publication C29-A2—Standardization of Sodium and Potassium Ion-Selective Electrode Systems to the Flame Photometric Reference Method; Approved Standard—Second Edition (ISBN 1-56238-410-4). Copies of the current edition may be obtained from NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Permission to reproduce or otherwise use the text of this document to an extent that exceeds the exemptions granted here or under the Copyright Law must be obtained from NCCLS by written request.
To request such permission, address inquiries to the Executive Director, NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Copyright ©2000. The National Committee for Clinical Laboratory Standards.
Suggested Citation
(NCCLS. Standardization of Sodium and Potassium Ion-Selective Electrode Systems to the Flame Photometric Reference Method; Approved Standard—Second Edition. NCCLS document C29-A2 [ISBN 1-56238-410-4]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2000.)
Proposed Standard December 1989 Tentative Standard December 1992 Approved Standard March 1995
Approved Standard—Second Edition October 2000
ISBN 1-56238-410-4 ISSN 0273-3099
Committee Membership
Area Committee on Clinical Chemistry and Toxicology Basil T. Doumas, Ph.D.
Chairholder Medical College of Wisconsin
Milwaukee, Wisconsin W. Gregory Miller, Ph.D.
Vice-Chairholder Virginia Commonwealth University
Richmond, Virginia
Paul D’Orazio, Ph.D. Instrumentation Laboratory
Lexington, Massachusetts
John H. Eckfeldt, M.D., Ph.D. Fairview-University Medical Center Minneapolis, Minnesota
Susan A. Evans, Ph.D. Dade Behring Inc.
Deerfield, Illinois
Gary A. Graham, Ph.D., DABCC Ortho-Clinical Diagnostics Rochester, New York
Gary L. Myers, Ph.D. Centers for Disease Control and Prevention Atlanta, Georgia
Patrick J. Parsons, Ph.D. New York State Department of Health Albany, New York
Noel V. Stanton, M.S. University of Wisconsin
Madison, Wisconsin Advisors
Judith T. Barr, Sc.D. Northeastern University
Boston, Massachusetts
Stanley Bauer, M.D. Beth Israel Medical Center
New York, New York George N. Bowers, Jr., M.D. Hartford Hospital
Hartford, Connecticut
Robert W. Burnett, Ph.D. Hartford Hospital
Hartford, Connecticut
Mary F. Burritt, Ph.D. Mayo Clinic
Rochester, Minnesota
Kevin D. Fallon, Ph.D. Instrumentation Laboratory
Lexington, Massachusetts
vi
Advisors (Continued)
Carl C. Garber, Ph.D. Quest Diagnostics, Incorporated
Teterboro, New Jersey
Harvey W. Kaufman, M.D. Quest Diagnostics, Incorporated Teterboro, New Jersey
Jan S. Krouwer, Ph.D. Bayer Diagnostics
Medfield, Massachusetts
Victoria M. Leitz, Ph.D. International Biomedical Consultants Hilton Head, South Carolina
Richard R. Miller, Jr. Dade Behring Inc.
Newark, Delaware Robert F. Moran, Ph.D., FCCM, FAIC mvi Sciences
Methuen, Massachusetts
Richard B. Passey, Ph.D. University of Oklahoma
Oklahoma City, Oklahoma
Edward A. Sasse, Ph.D. Medical College of Wisconsin
Milwaukee, Wisconsin Richard S. Schifreen, Ph.D. Promega Corporation
Madison, Wisconsin
Bette Seamonds, Ph.D. National Academy of Clinical Biochemistry Swarthmore, Pennsylvania
Beth Ann Wise, M.T.(ASCP), M.S.Ed.
Staff Liaison NCCLS
Wayne, Pennsylvania Patrice E. Polgar
Editor NCCLS
Wayne, Pennsylvania Donna M. Wilhelm
Assistant Editor NCCLS
Wayne, Pennsylvania
Acknowledgements
The Area Committee on Clinical Chemistry and Toxicology extends its appreciation to Paul D’Orazio, Ph.D., Co-Chairholder of the former Subcommittee on Electrolytes, for his help in preparing the second edition of this approved-level guideline, especially his advice on appropriate revisions and responses to the comments.
In addition, the area committee would also like to recognize the valuable contributions of the members and advisors of the Subcommittee on Electrolytes that developed the first approved edition of this guideline.
Paul D’Orazio, Ph.D., Co-Chairholder Gary Graham, Ph.D., Co-Chairholder Carolyn Bergkuist, M.S.
Ioannis Laios, Ph.D.
Alan Cormier, Ph.D.
Sharon Ehrmeyer, Ph.D.
Arthur Malenfant, Ph.D.
Richard R. Miller, Jr.
John Toffaletti, Ph.D.
Jesper Wandrup, M.D.
Advisors
George Bowers, Jr., M.D.
Richard A. Durst, Ph.D.
Neil Greenberg, Ph.D.
Jack Ladenson, Ph.D.
Robert F. Moran, M.S., Ph.D., FCCM, FAIC Kathleen M. O'Connell, Ph.D.
James Seago, Ph.D.
Paul Van Dreal, Ph.D.
viii
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OFFICERS BOARD OF DIRECTORS
F. Alan Andersen, Ph.D., President
Cosmetic Ingredient Review Donna M. Meyer, Ph.D., President Elect
CHRISTUS Health
Robert F. Moran, Ph.D., FCCM, FAIC
Secretary mvi Sciences
Gerald A. Hoeltge, M.D.
Treasurer
The Cleveland Clinic Foundation William F. Koch, Ph.D.,
Immediate Past President National Institute of Standards and Technology
John V. Bergen, Ph.D., Executive Director
Susan Blonshine, RRT, RPFT, FAARC
TechEd
Kurt H. Davis, FCSMLS, CAE Canadian Society for Medical Laboratory Science
Robert L. Habig, Ph.D.
Cytometrics, Inc.
Thomas L. Hearn, Ph.D.
Centers for Disease Control and Prevention
Elizabeth D. Jacobson, Ph.D.
Food and Drug Administration Carolyn D. Jones, J.D., M.P.H.
Health Industry Manufacturers Association
Tadashi Kawai, M.D., Ph.D.
International Clinical Pathology Center
J. Stephen Kroger, M.D., FACP COLA
Barbara G. Painter, Ph.D.
Bayer Corporation Emil Voelkert, Ph.D.
Roche Diagnostics GmbH Ann M. Willey, Ph.D.
New York State Department of Health
Judith A. Yost, M.A., M.T.(ASCP) Health Care Financing
Administration
xiv
Contents
Abstract...i
Committee Membership...v
Active Membership ...ix
Foreword...xvii
1 Introduction...1
2 Scope...1
3 Definitions/Terminology...1
3.1 Abbreviations...2
4 Standard Precautions...2
5 Purpose...2
5.1 Whole Blood and Undiluted Plasma or Serum...3
5.2 "Normal" Specimens...3
5.3 Characteristics of a "Normal" Specimen...3
6 Background...3
6.1 Preanalytical Effects on Patient Sample Results: Influences of Specimen Choice, Collection, and Handling...3
6.2 Analytical Issues...4
6.3 Reporting Results...5
7 Standardization of Result Reporting...5
7.1 Materials...5
7.2 Sample Analysis and Data Reduction...6
7.3 Validation Protocol Using Patient Samples (Optional)...7
References...8
Appendix A. Specifications for a Human Serum Certified Reference Material (CRM) for the ISE Measurement of Sodium and Potassium in Blood...10
Appendix B. Estimated Number of Replicates for Demonstrating Equality of Flame and ISE Determination...13
Appendix C. An Example of Normalization of Linear Regression Data for Sodium to 140 mmol/L and Adjustment of the Slope to One and Intercept to Zero...14
Appendix D. Determination of Plasma Water Mass Concentration...16
Summary of Comments and Subcommittee Responses...17
Summary of Delegate Comments and Responses...18
Related NCCLS Publications...21
xvi
Foreword
Analysis of electrolytes in whole blood is becoming increasingly common, and instruments that perform these analyses (ion-selective electrodes that do not require diluted samples, i.e., direct ISE) are available from a number of manufacturers. In many clinical laboratories, instruments that use a diluted specimen [flame photometry or indirect ion-selective electrodes (ISEs)] are also still in use. Direct ISE results are not equivalent to those results obtained by the technology employing dilution for a number of reasons, foremost of which are those relating to the variation in plasma water. To avoid confusion, we recommend that results obtained by direct potentiometry be adjusted to resemble those obtained by procedures that measure concentration in plasma. Most instruments using direct ISEs have built-in conversion algorithms that can be utilized by putting the instrument in "flame" mode. However, it is clear from work performed at the National Institute of Standards and Technology (NIST) that a number of direct potentiometric systems from a variety of manufacturers do not give identical results when assaying identical specimens even in the "flame" mode.1
The results of determinations of sodium and potassium ions in physiological fluids have been expressed in terms of (substance) concentration (mmol/L) for many years. The use of concentrations of both sodium and potassium and their reference intervals is firmly established in clinical interpretation and practice. Analytical systems that report concentration, such as flame atomic emission spectrometry (FAES) and ion-selective electrode systems using diluted samples, will continue to be used alongside direct ion-selective electrode determinations in the foreseeable future. Consequently, to introduce a new system of reporting results of sodium and potassium determinations by ion-selective electrodes in terms of ion activity carries significant risks of confusing clinical interpretation.2,3
A more fundamental problem also exists. Ion-selective electrodes respond to the thermodynamic activity of ions in solution. By theory, they do not sense concentration which, in fluids such as plasma, is related in a complex way to activity.4, 2 Therefore, results should be expressed in terms of ion activity. Many practical difficulties exist with this approach, however. Because the activity of these ions cannot, at present, be determined with certainty, particularly in a fluid as complex as plasma, the accuracy of any determination of activity cannot be verified.
The convention recommended in this standard represents a pragmatic compromise that facilitates the introduction of ion-selective electrode determinations of sodium and potassium ion concentrations in whole blood or undiluted plasma into routine clinical practice, while minimizing the risk of clinical misinterpretation.
Key Words
Direct potentiometry, flame photometry, ion-selective electrode (ISE), potassium, sodium
xviii
An NCCLS global consensus standard. ©NCCLS. All rights reserved.
Standardization of Sodium and Potassium Ion-Selective Electrode Systems to the Flame Photometric Reference Method; Approved Standard—
Second Edition 1 Introduction
The objective of this standard is to describe a method to standardize direct ion-selective electrode analyzers for determination of sodium and potassium in blood plasma to units of concentration (mmoI/L) in “normal” plasma, as reported by flame atomic emission spectrometry (flame photometry). This standardized method will allow clinical laboratories to use the same reference intervals, regardless of instrumentation or principle of the methodology.
2 Scope
This document addresses the determination of sodium and potassium in undiluted plasma in routine clinical practice using ion-selective electrodes.
3 Definitions/Terminology
aCalibration material//Calibrator, n - A material or device of known, or assigned quantitative characteristics (e.g., concentration, activity, intensity, reactivity, responsiveness) used to adjust the output of a measurement procedure or to compare the response obtained with the response of a test specimen and/or sample.
Direct analysis, n - Measurement made directly on an undiluted specimen, e.g., whole blood, plasma, or sweat.
Flame mode, n - In the flame mode, a factor can be applied to results generated by direct ISE systems that makes the results comparable to those generated by indirect systems for patient specimens normal in protein and lipid content.
Heparinized, adj - Specimens anticoagulated with a heparin salt(s).
Indirect analysis, n - Systems that require dilution of the sample; NOTE: These include some ion-selective, electrode-based systems, as well as flame emission photometry and atomic absorption.
Plasma, n - The liquid part {of whole blood} remaining after the separation of the cellular elements ... in a receptacle containing an anticoagulant, or separated by continuous filtration or centrifugation of anticoagulated blood in an apheresis procedure.
Primary standard, n - A standard that is designated or widely acknowledged as having the highest metrological qualities and whose value is accepted without reference to other standards of the same quantity.
a Some of these definitions are found in NCCLS document NRSCL8C Terminology and Definitions for Use in NCCLS Documents. For complete definitions and detailed source information, please refer to the most current edition of that document.
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2
3.1 Abbreviations
Sectionb
ACD Acid citrate dextrose A2.1
CRM/SRM Certified reference material/standard reference material 7.1/App. A
CV Coefficient of variation 7.2.2
EP-BGpH/IFCC Expert Panel on Blood Gas/pH of the IFCC A3
FAAS Flame atomic absorption spectrometry A2.3
FAES Flame atomic emission spectroscopy A2.1(4)
FP Flame photometer 6.2.2
HIV Human immunodeficiency virus A2.1
IFCC International Federation of Clinical Chemistry A3
ISE Ion-selective electrode 3
NIST National Institute of Standards and Technology 7.1
NRSCL National Reference System for the Clinical Laboratory A1.3
4 Standard Precautions
Because it is often impossible to know what might be infectious, all human blood specimens are to be treated as infectious and handled according to “standard precautions.” Standard precautions are new guidelines that combine the major features of “universal precautions and body substance isolation”
practices. Standard precautions cover the transmission of any pathogen and thus are more comprehensive than universal precautions which are intended to apply only to transmission of blood-borne pathogens.
Standard precaution and universal precaution guidelines are available from the U.S. Centers for Disease Control and Prevention (Guideline for Isolation Precautions in Hospitals. Infection Control and Hospital Epidemiology. CDC. 1996;Vol 17;1:53-80.), [MMWR 1987;36(suppl 2S):2S-18S] and (MMWR 1988;37:377-382, 387-388). For specific precautions for preventing the laboratory transmission of blood- borne infection from laboratory instruments and materials; and recommendations for the management of blood-borne exposure, refer to NCCLS document M29—Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue.
5 Purpose
The purpose of this standard is to recommend a procedure for the standardization of reported, direct ion- selective electrode determination of sodium and potassium in serum, plasma, or whole blood.
bSection where abbreviation first appears in text.
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A convention for reporting results of ion-selective electrode determinations of sodium and potassium is proposed whereby, for routine clinical purposes:
5.1 Whole Blood and Undiluted Plasma or Serum
Results of ion-selective electrode determinations of sodium and potassium in whole blood and undiluted
5.2 "Normal" Specimens
Results of measurements on "normal" specimens should conform with those obtained by flame atomic emission spectrometry on the same specimens.
5.3 Characteristics of a "Normal" Specimen
"Normal" plasma specimens are herein defined as having mass concentration of plasma water of 0.93 ± 0.01 kg/L; plasma total CO2 of 24 ± 2 mmol/L; plasma pH of 7.40 ± 0.05; and concentrations of albumin, total protein, cholesterol, and triglycerides as specified in Section 7.3.
6 Background
6.1 Preanalytical Effects on Patient Sample Results: Influences of Specimen Choice, Collection, and Handling
Electrolyte results, especially for potassium, may be affected by cellular transfer that occurs either during patient preparation, sample collection, or sample handling.
Because the sodium concentration in erythrocytes is one-tenth that of plasma, hemolysis has little effect on the sodium concentration. For example, hemolysis resulting in a plasma hemoglobin of 5 g/L would decrease a 140-mmol/L plasma sodium concentration by only 0.4%. Sodium concentrations are markedly affected only by extremely severe (>10 g/L Hb) hemolysis.
Because the intracellular potassium concentration is about 23 times higher than that in plasma, cellular disruption or cellular leakage of potassium can markedly alter the potassium concentration of plasma.
6.1.1 Cellular Disruption
For example, hemolysis resulting in a plasma hemoglobin of 5 g/L would increase plasma potassium concentration by 0.5 mmol/L. Because platelets are ruptured during coagulation, serum potassium is increased by 0.1 to 0.7 mmol/L, or even higher, in patients with thrombocytosis.5 Therefore, plasma is preferred for determination of potassium.6
6.1.2 Cellular Leakage
Potassium movement through intact cell membranes can also alter plasma potassium by several mechanisms during specimen collection.
6.1.2.1 Extracellular Acidosis
Extracellular acidosis due to tourniquet application with forearm exertion leads to exchange of hydrogen ion for potassium resulting in a 10 to 20% increase in plasma potassium.7 The tourniquet should not be released until the collection is completed.
plasma or serum (see Section 3) should be reported in terms of concentration (mmol/L).
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4
6.1.2.2 Intracellular Glucose Depletion
Intracellular glucose depletion by glycolysis induces glucose entry into cells. This entry is accompanied by potassium, decreasing extracellular potassium. This mechanism is an early and limited process, especially evident in patients with leukocytosis (leukocyte count above 100 x 109/L).
6.1.2.3 Sodium/Potassium Pump
Cellular leakage is dependent upon the sodium/potassium pump. Its efficiency is temperature- and energy-dependent. When temperature decreases, the pump slows down, causing the potassium to leak out of the cell. The rise in plasma potassium is about 0.15 mmol/L per hour at 25"C and about 0.25 mmol/L per hour at 4"C6 (for example, after storage of a syringe in ice water). Some anticoagulants, which inhibit glycolysis, can also increase plasma potassium.6
6.1.3 Collection and Handling
To summarize the practical recommendations for collection and handling:
• Prolonged tourniquet application (in excess of 2 minutes) and forearm exertion by the patient should be avoided.
• Blood should be collected with Li heparin as an anticoagulant.
• Samples should be kept at room temperature (20 to 25"C).
• Plasma should be separated from cells within 60 minutes after collection.
6.2 Analytical Issues
6.2.1 Direct Measurement
In a properly designed direct ion-selective electrode system, differences in sodium and potassium measurements between heparinized plasma and whole blood should be minimal. The differences seen are attributed primarily to changes in the liquid-junction potential due to the erythrocytes and can be minimized with the use of properly designed systems and electrolyte solutions to form the liquid junction.8
6.2.2 Indirect Methods
The traditional methods measure the concentration of Na, K in plasma (e.g., FP and "diluting" ISE instruments.) The FP measures the intensity of light emitted by the sodium and potassium atoms excited by the flame. This light intensity is directly proportional to the number of sodium and potassium atoms, which, in turn, is directly proportional to the concentration of these ions in the sample. Instruments using diluted ISE specimens also measure the plasma sodium/potassium concentration, because the sample is diluted such that both the activity coefficient of sodium and potassium and the volume fraction of water in the sample is nearly equal to the activity coefficient of sodium and potassium and the volume fraction of water in the calibrating solutions. Under these conditions, activity is directly proportional to concentration with the same proportionality constant from sample to sample.
6.2.3 Analytical Comparison: Direct versus Indirect
About 47% of the participants in the 1994 C-A Comprehensive Chemistry Survey of the College of American Pathologists9 used diluted ISE methods for determining sodium. Another 1% of the
An NCCLS global consensus standard. ©NCCLS. All rights reserved.
participants used the FP. Diluted ISE methods are referred to as "indirect" methods. The difference in the sodium and potassium results between the direct and indirect methods is mainly due to the electrolyte
6
plasma. Sodium ions are almost exclusively associated with the water phase of plasma. Because the volume fraction of plasma water in normal specimens is approximately 93%, indirect ISEs and FPs will produce results that are about 7% lower than direct ISE methods. All methods that measure the concentration of sodium in plasma are related to the total volume of sample and not the water phase of that sample. As the volume fraction of plasma water is altered by disease, the difference between the direct and indirect methods will also change (see Section 6.2.4). Another source of differences between direct and indirect methods is the binding of these ions by inorganic and organic ligands, primarily bicarbonate. This binding is minimal (1 to 2%) as compared to the influence of plasma water (7%).
6.2.4 Abnormal Specimens
In all samples with abnormal lipid and protein levels, diluted methodologies give results that are biased from measurements on undiluted samples. For specimens with abnormal concentrations of plasma water, bicarbonate, or hydrogen ions, the results reported by ion-selective electrodes on undiluted samples and by flame atomic emission spectrometry diverge. Results obtained by ion-selective electrodes on undiluted samples more accurately reflect the pathophysiological status of these ions in plasma water and thus may be more relevant clinically than those reported by flame atomic emission spectrometry.11,12 Difference in sodium values due to protein concentration can be as high as 17 mmol/L (total protein ~160 g/L),13 while differences due to lipid concentration (triglyceride ~113,000 mg/L) were reported to be 26 mmol/L.14
6.3 Reporting Results
If results by both techniques are available, it should be made clear whether they are derived from measurements with ISEs on undiluted specimens or with diluted specimens by ISE or FP. This is necessary, because certain samples with abnormalities in lipid, protein, or bicarbonate concentration can give clinically important differences in electrolytes depending on the analytical procedure. The risk for clinical misinterpretation is present if results from ISE using sample dilution or FP only are reported for samples with these abnormalities. In these cases, the direct ISE result is to be regarded as the more clinically relevant result.
7 Standardization of Result Reporting
The objective of this standard is to describe a method to standardize direct ion-selective electrode analyzers for determination of sodium and potassium in blood plasma to units of concentration (mmol/L) in "normal" plasma, as reported by flame atomic emission spectrometry (flame photometry). Bias of the ISE analyzer to flame photometry after this standardization process should be within ±2%. This is the maximum allowable population difference, irrespective of the sodium or potassium concentration, within the 120- to 160-mmol sodium/L or 2- to 6-mmol potassium/L plasma ranges. Therefore, clinical laboratories will be able to use the same reference intervals, regardless of instrumentation or principle of the methodology.
7.1 Materials
Three serum pools should be included in this method. Three reference materials, formulated for the purpose of standardizing ion-selective electrode analyzers to flame photometry, are available from NIST [Gaithersburg, MD; telephone (301) 975-OSRM] and are recommended (SRM 956a). The sodium and potassium concentrations (mmol/L) of these pools are as follows:
10
exclusion effect. This effect is caused by the solvent-displacing effect of lipids and proteins in the
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6
Level 1 Level 2 Level 3
Sodium 120 ± 2 140 ± 2 160 ± 2
Potassium 6.0 ± 0.2 4.0 ± 0.2 2.0 ± 0.2
As an alternative to purchasing these materials, the serum pools may be prepared using the method described in Appendix A. Alternative materials from other countries may be acceptable upon verification by the user according to specifications listed in Appendix A.
7.2 Sample Analysis and Data Reduction
The following protocol may be used with SRM 956a or with serum pools. If SRM 956a is used, then the label values assigned by NIST should be used below in lieu of the flame photometric concentrations. The Subcommittee on Electrolytes believes that the most practical method for standardization and/or verification of the standardization of ISE instruments is to use the NIST standard reference material.
7.2.1 Calibration and Analysis
The ISE instrument should be calibrated according to the manufacturer's instructions and operated in the flame mode when this option is available. The FP should be calibrated according to the NIST reference method.15,16 Each of the three serum pools should be analyzed by alternating determinations on the ISE analyzer and FP. They should be run in random order with respect to concentration, for a total of 15 replicates on each instrument for each concentration.
Example: Individual determinations might be sequenced as:
(ISE1 FP1 FP3 ISE3 ISE2 FP2) (FP3 ISE3 ISE1 FP1 ISE2 FP2), etc.
where: the numbers refer to analyte level and the parentheses enclose a complete set of replicates across instruments and levels.
7.2.2 Coefficient of Variation (CV)
For this protocol to be valid, the coefficient of variation (%CV, within-run) should be less than or equal to 1.5% at all levels on both instruments (see Appendix B). If this level of imprecision is not obtained, consult the instrument operating manuals for the appropriate troubleshooting procedures, or consult the manufacturer. The means of the determinations from the FP and the ISE analyzer should agree to within
±1% for both Na+ and K+ at all concentrations. This will guarantee, at the 95% level of confidence, that the true difference between flame and ISE is within ±2%.
If ±1% agreement is not obtained above, then the results for the two methods should be analyzed by linear regression following normalization to 140 and 4.00 mmol/L Na+ and K+, respectively (subtraction of 140 and 4.00 from all mean values). The best fit lines for Na+ and K+ should then be adjusted to a slope of 1 and an intercept of 0 using the appropriate correction factors (see Appendix C for an example of this procedure.)
7.2.3 Slope and Intercept Correction Factors
Certain commercial analyzers allow the slope and intercept correction factors to be entered into the instrument's microprocessor at the user level. If this feature is not available, the corrections to all subsequent data can be done manually or through an appropriate data management system.
17
An NCCLS global consensus standard. ©NCCLS. All rights reserved.
7.3 Validation Protocol Using Patient Samples (Optional)
If desired, patient sera may be used to test the above procedure either immediately following standardization or at a future time to determine if the procedure should be repeated. These specimens should be no more than 24 hours old, maintained in an anaerobic state, and known to be within the normal ranges for the following analytes18:
Table 1. Normal Analyte Ranges for Adults
Analyte Method Mean Limits Units
Total CO2 volumetric 25 22 to 29 mmol/L
Protein, total NRS/TP 70 63 to 79 g/L
Albumin rapid BCG 45 35 to 50 g/L
Cholesterol NRS/CHOL 2,000 1,500 to 2,500 mg/L
Triglycerides Enz/Blank 1,000 500 to 1,500 mg/L
pH (at 37 °C) glass ISE 7.40 7.35 to 7.45
Calibration of the ISE analyzer and the FP should be as described in Section 7.2. To accurately test the standardization function, a minimum of 30 patient samples chosen to span the analytical range of Na+ (120 to 160 mmol/L) and K+ (2.0 to 6.0 mmol/L) as closely as possible should be run in duplicate on each instrument. The duplication is necessary to improve the precision of the estimate and to detect outliers that may be due to sample handling. Evaluate outliers according to standard techniques and repeat the sample analysis. The data should then be normalized to 140 and 4.0 mmol/L Na+ and K+, respectively (Section 7.2).
Regression analysis on the normalized data should be performed pairing the first ISE duplicate with the first FP duplicate. The ISE instrument value should be used as the "y" or dependent variable. The slope estimate for each analyte should be within the 0.95 to 1.05 interval. The intercept should be within the interval -1 to +1 for Na+ and -0.03 to +0.03 for K+. If these criteria are not met, then the standardization procedure of Sections 7.1 and 7.2 should be repeated.
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8
References
1 Gunaratna PC, Paule RC, Koch WF. Development of reference materials for potentiometric sodium and potassium measurements in serum. In Burritt MF et al., eds. Methodology and Clinical Application of Ion-Selective Electrodes. American Association for Clinical Chemistry/International Federation for Clinical Chemistry, 1987:52-66.
2 Buckley BM et al. New ways with old ions. Ann Clin Biochem. 1984;21:75.
3 Sachs C, Truchaud A. Reference intervals for plasma electrolyte measurements: what is realistic and acceptable in routine clinical diagnosis and therapy in 1983? In Koch WF, ed. Proceedings of the Workshop on Direct Potentiometric Measurement in Blood. Washington, DC: The National Committee for Clinical Laboratory Standards and the National Bureau of Standards. 1985:182-184.
4 Maas AHJ et al. Ion-selective electrodes for sodium and potassium: A new problem of what is measured and what is reported. Clin Chem. 1985;31:482-485.
5 Ingram RH, Seki M. Pseudohyperkalemia with thrombocytosis. N Eng J Med. 1962;267:895-900.
6 Tietz NW, Pruden EL, Siggaard-Andersen O. Electrolytes, blood gases, and acid base balance. In Tietz NW, ed. Textbook of Clinical Chemistry. Philadelphia: WB Saunders. 1986:1174-1177.
7 Renoe BW, McDonald JM, Ladenson JH. The effects of stasis with and without exercise on free calcium, various cations, and related parameters. Clin Chim Acta. 1980;103:91-100.
8 Meyerhoff ME, Opdycke WN. Ion-selective electrodes. In Spiegel HE, ed. Advances in Clinical Chemistry. New York: Academic Press, Inc. 1986:1-47.
9 College of American Pathologists. Set C-A Comprehensive Chemistry Survey Report. Chicago:
CAP; 1994.
10 Wimberley PD et al. Are sodium bicarbonate and potassium bicarbonate fully dissociated under physiological conditions? Scand J Clin Lab Invest. 1985;45:7-10.
11 Burn J, Gill GV. Pseudonormonatraemia. Br Med J. 1979:1110-1111.
12 Frier BM et al. Misleading plasma electrolytes in diabetic children with severe hyperlipidaemia.
Arch Dis Child. 1985;55:771-775.
13 Ladenson JH et al. Sodium measurements in multiple myeloma: Two techniques compared. Clin Chem. 1982;28:2383-2386.
14 Ladenson JH, Apple FS, Koch DD. Misleading hyponatraemia due to hyperlipemia: A method-dependent error. Ann Intern Med. 1981;95:707-708.
15 Velapoldi RA et al. A Reference Method for the Determination of Sodium in Serum. National Bureau of Standards Special Publication 260-262. Washington, DC: US Government Printing Office; 1978.
16 Velapoldi RA et al. A Reference Method for the Determination of Potassium in Serum. National Bureau of Standards Special Publication 260-263. Washington, DC: US Government Printing Office;
1979.
An NCCLS global consensus standard. ©NCCLS. All rights reserved.
References (Continued)
17 Dixon WJ, Massey FJ. Introduction to Statistical Analysis. 4th ed. New York: McGraw Hill.
1983:564.
18 Tietz NW. Clinical Guide to Laboratory Tests. Philadelphia: WB Saunders; 1983.
10 An NCCLS global consensus standard. ©NCCLS. All rights reserved.
Appendix A. Specifications for a Human Serum Certified Reference Material (CRM) for the ISE Measurement of Sodium and Potassium in Blood
This CRM is specifically designed by the Electrolyte Working Group (EWG) to meet the standardization needs that are unique to the direct ISE potentiometric measurement of sodium and potassium in undiluted human serum or plasma. The substance concentrations at the "low," "mid," and "high" levels are to be directly traceable to the National Reference System for Sodium (NRS/NA)1and Potassium (NRS/K)2 by value assignment with the definitive methods at NIST. Therefore, this CRM should unify the results for Na and K on human serum, plasma, and whole blood (whether made on undiluted or diluted samples) on almost any analytical system.
A1 Manufacturing Procedures
A1.1 Records, Documentation, and Reports
Pay careful attention to records, documentation, and reports; freedom from chemical and bacterial contaminants; and analytical methods and traceability.
Complete records on the source and specifications for all materials, including dates received, and the criteria for acceptance. Also, maintain a log of all procedures and processing steps.
A1.2 Freedom from Chemical and Bacterial Contaminants
Employ clean-room-type manufacturing practices to reduce particulate and bacterial contamination. Use chemically clean vats, mixing tanks, and apparatus in the manufacture of this CRM to avoid contamination from any carry-over of trace materials from prior manufacturing activities.
A1.3 Analytical Methods and Traceability
The accuracy of analytical methods used and of the results obtained should be traceable to the analyte summaries of the NRSCL/NCCLS. When a certified NIST/SRM for an analyte and/or serum reference materials is available, it should be run concurrently to help validate the methods and their results.
A2 Starting Material and Preparation of This CRM
A2.1 Starting Material, Biohazards, and Base Pool Specifications
Only native human serum (not ACD plasma) that has been tested and found negative for HIV and hepatitis is to be used. The values of sodium, potassium, and the other analytes listed in Table A1 (in this appendix) in the pooled sera should be within the usual reference intervals for a "healthy" adult population.
Tonometry of this pool with CO2 gas should adjust the pH and pCO2 to approximately the mean of normal.
(1) Freeze the serum pool (-20 °C) and thaw it; a visible cloudiness (presumably lipoproteins) should be observed. If cloudiness is not observed, repeat the freeze-thaw cycle.
(2) Filter the base pool through an Avicel cellulose slurry under a vacuum (see Dujardin BCG, Roijers AFM. Preparation of an optically clear frozen human control serum. Scand J Clin Lab Invest. 1985;45:569-572), or equivalent clearing process to reduce turbidity. The absorbance with a 1-cm path length at 700 nm should be less than 0.1. This treatment also helps to reduce the
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Appendix A. (Continued)
rate of clogging in subsequent finer nitrocellulose filtration steps that help to ensure the homogeneity and reduce the bacteria count.
(3) After the above clearing step, add gentamicin sulfate and mix it into the base pool to give a final concentration of 50 mg/L. The addition of gentamicin sulfate is necessary if long-term storage of sera is anticipated. Gentamicin may be omitted if samples are to be stored for one week or less.
In this case, storage should be in capped glass containers under 5% CO2, with no more than 5 mL per container (no more than 100% head space), and frozen at -20 °C. The pool is filtered through a coarse prefilter and then progressively down through various pore-sized nitrocellulose filters as follows: 3.00, 1.20, 0.80, 0.65, 0.45, 0.30, and finally 0.22 mm.
(4) Remove a small aliquot of this filtered base, and measure it for the potassium concentration by FAES and the total protein by the Biuret reaction. Store another 20-mL aliquot (4 "C) for future simultaneous comparison of analyte levels versus the new "low," "mid," and "high" CRMs.
A2.2 Dilution and Ultrafiltration of the Base Pool For this procedure:
(1) The filtered base pool should be diluted with a sodium bicarbonate (25 mmol/L) solution so that the resulting potassium is 2.0 ± 0.05 mmol/L by FAES and the total CO2 is 25 ± 1 mmol/L by volumetric technique.
(2) The diluted base pool should be ultrafiltered at 6 ± 3 °C until the total protein of the retentate reaches 70 ± 2 g/L using an appropriately sized apparatus and a filter of 20,000 d cutoff size. The retentate should maintain a pH of 7.4 ± 0.05 and the bicarbonate of 24 mmol/L. All pH measurements should be made at 37 °C. The pH of the retentate should be adjusted to 7.4 using 5% CO2 gas.
A2.3 Adjustments to the "Low" and "High"
The appropriate amounts of chloride salts (autoclaved pools or filtered through 0.22 µm) should be added to each subpool to give the following final mmol/L substance concentrations (Na/K by FAES and Ca/Mg by FAAS):
Na (1) K (2) Ca Mg Li Ionic S
"Low" Na subpool 120 ± 3 6.0 ± 0.2 3.0 ± 0.2 1.5 ± 0.1 2.0 ± 0.1 (145)
"High" Na subpool 160 ± 3 2.0 ± 0.2 2.0 ± 0.2 0.5 ± 0.1 0.5 ± 0.1 (175) A2.4 “Mid” Na Subpool Preparation
"Mid" Na subpool is made from equal amounts of "low" and "high" and, in theory, should give values close to the average, as shown below:
Na (1) K (2) Ca Mg Li Ionic S
"Mid" Na subpool 140 ± 3 4.0 ± 0.2 2.5 ± 0.2 1.0 ± 0.1 1.2 ± 50.1 (160)
12 An NCCLS global consensus standard. ©NCCLS. All rights reserved.
A2.5 Final Filtration Dispensing, Packaging, and Storage
(1) Refilter each of these three subpools through a pore size of 0.22 µm to better ensure the homogeneity and further reduction in the number of microorganisms. Adjust the pH to 7.40
± 0.05 using 5% CO2 gas.
(2) Dispense 3.0-mL aliquots of each subpool into 5-mL glass ampules flushed with and sealed under an inert gas plus 5% CO2. Store the processed CRM immediately at -50 °C and transport it under dry ice.
A3 Value Assignment, Unit Size, and Distribution
The responsibility for the final value assignment and certification of this CRM rests with NIST.
Definitive method values traceable to the NRSCL/NCCLS should be used.
A packaged unit of this CRM is six frozen ampules (two "low," two "mid," and two "high") shipped under dry ice. Distribution is the sole responsibility of the Standard Reference Materials Program at NIST (SRM 956a).
NOTE:The ionic strength of each of these three subpools is different and only the "mid" subpool is about 160 mmol/kg of water, as recommended by the EP-BGpH/IFCC.
Table A1. Base Pool Limits (See Section A2.1)
Analyte Method Mean and Limits, Adults17 Units
Sodium FAES 141 136 to 146 mmol/L
Potassium FAES 4.2 3.5 to 5.1 mmol/L
Calcium FAAS 2.4 2.1 to 2.5 mmol/L
Magnesium FAAS 0.9 0.7 to 1.1 mmol/L
Lithium FAAS 0.0 0.0 to 0.0 mmol/L
Chloride NRS/C1 102 98 to 106 mmol/L
Total CO2 Volumetric 25 22 to 29 mmol/L
Protein, total NRS/TP 70 63 to 79 g/L
Albumin rapid BCG 45 35 to 50 g/L
Cholesterol NRS/CHOL 2,000 1,500 to 2,500 mg/L
Triglyceride Enz/blank 1,000 500 to 1,500 mg/L
Urea nitrogen NRS/UREA 100 70 to 210 mg/L
Creatinine Jaffe/Enz 8 5 to 12 mg/L
Ammonia Enzymatic 25 11 to 35 µmol/L
References to Appendix A.
1. National Committee for Clinical Laboratory Standards. Potassium; Proposed Summary of Methods and Materials Credentialed by the NRSCL Council. NCCLS Document RS8-P. NCCLS, 771 East Lancaster Avenue, Villanova, Pennsylvania 19085; 1988.
2. National Committee for Clinical Laboratory Standards. Sodium; Proposed Summary of Methods and Materials Credentialed by the NRSCL Council. NCCLS Document RS7-P. NCCLS, 771 East Lancaster Avenue, Villanova, Pennsylvania 19085; 1988.
