NBS SPECIAL PUBLICATION 260*36

Standard Reference Materials:

A REFEREE METHOD

FOR THE DETERMINATION OF

CALCIUM IN SERUM
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards1 was established by an act of Congress March 3, '901. The Bureau's overall goal is to strengthen and advance the Nation's science and

technology and facilitate their effective application for public benefit. To this end, the Bureau conducts research and provides: (1) a basis for the Nation's physical measure- ment system, (2) scientific and technological services for industry and government, (3) a technical basis for equity in trade, and (4) technical services to promote public safety. The Bureau consists of the Institute for Basic Standards, the Institute for Materials Research, the Institute for Applied Technology, the Center for Computer Sciences and

Technology, and the Office for Information Programs. THE INSTITUTE FOR BASIC STANDARDS provides the central basis within the United States of a complete and consistent system of physical measurement; coordinates

that system with measurement systems of other nations; and furnishes essential services leading to accurate and uniform physical measurements throughout the Nation's scientific community, industry, and commerce. The Institute consists of a Center for Radiation Research, an Office of Measurement Services and the following divisions: Applied Mathematics—Electricity—Heat—Mechanics—Optical Physics—Linac

Radiation2—Nuclear Radiation2—Applied Radiation2—Quantum Electronics3—

Electromagnetics3—Time and Frequency3 —Laboratory Astrophysics3—Cryogenics3

. THE INSTITUTE FOR MATERIALS RESEARCH conducts materials research lead- ing to improved methods of measurement, standards, and data on the properties of

well-characterized materials needed by industry, commerce, educational institutions, and

Government; provides advisory and research services to other Government agencies; and develops, produces, and distributes standard reference materials. The Institute consists of the Office of Standard Reference Materials and the following divisions: Analytical Chemistry—Polymers—Metallurgy—Inorganic Materials—Reactor

Radiation—Physical Chemistry. THE INSTITUTE FOR APPLIED TECHNOLOGY provides technical services to pro- mote the use of available technology and to facilitate technological innovation in industry and Government; cooperates with public and private organizations leading to the development of technological standards (including mandatory safety standards), codes and methods of test; and provides technical advice and services to Government agencies upon request. The Institute also monitors NBS engineering standards activities and

provides liaison between NBS and national and international engineering standards

bodies. The Institute consists of the following divisions and offices: Engineering Standards Services—Weights and Measures—Invention and

Innovation—Product Evaluation Technology—Building Research—Electronic Technology—Technical Analysis—Measurement Engineering—Office of Fire Programs. THE CENTER FOR COMPUTER SCIENCES AND TECHNOLOGY conducts re- search and provides technical services designed to aid Government agencies in improving cost effectiveness in the conduct of their programs through the selection, acquisition, and effective utilization of automatic data processing equipment; and serves as the principal focus within the executive branch for the development of Federal standards for automatic data processing equipment, techniques, and computer languages. The Center

consists of the following offices and divisions: Information Processing Standards—Computer Information—Computer Services —Systems Development—Information Processing Technology. THE OFFICE FOR INFORMATION PROGRAMS promotes optimum dissemination and accessibility of scientific information generated within NBS and other agencies of

the Federal Government; promotes the development of the National Standard Reference Data System and a system of information analysis centers dealing with the broader

aspects of the National Measurement System; provides appropriate services to ensure

that the NBS staff has optimum accessibility to the scientific information of the world, and directs the public information activities of the Bureau. The Office consists of the following organizational units: Office of Standard Reference Data—Office of Technical Information and

Publications—Library—Office of International Relations.

1 Headquarters and Laboratories at Gaithersburp, Maryland, unless otherwise noted; mailing address Washington, DC. 20234.

- Part of the Center for Radiation Research.

3 Located at Boulder, Colorado 80302.
ional Bureau of Standards

juai 2 7 1972 Standard Reference Materials:

[I V A Referee Method for the

Determination of Calcium in Serum

J. Paul Cali, John Mandel, and

Larry Moore

Institute for Materials Research

National Bureau of Standards Washington, D.C. 20234

and

Donald S. Young

National Institutes of Health Department of Health, Education and Welfare Washington, D.C. 20214

U.S. DEPARTMENT OF COMMERCE, Peter G. Peterson, Secretary

Issued May 1972
Library of Congress Catalog Card Number: 72-600082

National Bureau of Standards Special Publication 260-36

Nat. Bur. Stand. (U.S.), Spec. Publ. 260-36, 136 pages (May 1972) CODEN: XNBSAV

Issued May 1972

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 (Order by SD Catalog No. C13.10:260-36). Price $1.25.
Foreword

The "260 Series" of NBS Special Publications was originally initiated and reserved for the purpose of reporting

technical work done at NBS in the area of Standard Reference

Materials. The scope of this program was recently enlarged

to include support in the development of referee methods of

analysis, i.e., methods of known accuracy based in part on

the use of SRM 1 s . It is now recognized that a meaningful

measurement system comes into being only when its several

parts are brought into a harmonious and relevant relationship. The development of referee methods of analysis is a necessary step in this process. These matters are discussed

in detail in Sections I and II in the body of this report.

In Clinical Chemistry, referee methods, as defined

herein, are lacking. This work is a first attempt to bring

about a referee method of analysis in this field. A careful

study of the methodology actually used will reveal some shortcomings, but it may be said with some assurance,

subject to the many limitations and provisos listed, that

a referee method for the analysis of calcium in serum or physiological solutions has been achieved. It may be argued

that additional work to establish the magnitude of the several

individual systematic biases could have been carried out,

or that additional exercises should have been run, etc. However, when work of this nature is dependent to a large

degree upon the voluntary participation of the cooperating

laboratories and the free time and services of the Experts

Committee then there must be a reasonable limit as to the

total duration of the project. The work reported covered

the period from June 1970 to December 1971. Furthermore,

the Experts Committee felt that a reasonable goal had in

fact been achieved, based on the work to date, and that

further additional studies would not result in any marked

improvement in the accuracy of the method.

iii
Several of the findings are subjectively based and can- not be rigorously or fully supported by the cold evidence,

e.g., the requirement that "well-motivated personnel, trained

in the proper techniques are essential if the accuracy inherent in the method is to be achieved." We have no hesitancy in making such statements other than they are likely

to produce some rather heated controversy , because this work

is to be considered, in no small measure, as an educational

exercise. Automation in clinical chemistry, or any other

analytical field, cannot substitute for the technical skills

and knowledge necessary to the practice of analytical chemistry—and clinical chemistry, in practice, is clinical

analytical chemistry.

A word of caution is necessary. Referee methods have

been called, at various times and for various purposes,

standard methods , standard procedures , reference methods

,

validation methods, and a variety of other names. Because

the principal purpose is to achieve accuracy in measurement,

the term, referee method, is preferred over any other, to avoid implications of a legally imposed method, a preferred

method, a method of qualification and the like. Although

it is true that a referee method might, indeed, serve any one or more of these purposes, the firm intent of all con- cerned in developing the methodology described herein is

wholly scientific.

Finally, it should be understood that the "260 Series"

permits reporting in much more detail than is usually

allowable in scientific journals. Again, the reason for this

is largely educational. Very often in condensed versions

a great deal of expertise, experience, and technique is

assumed to reside in or be available to the reader. In this report we make a minimum of such assumptions . Further- more a degree of editorial comment and expounding of the

philosophical basis of the work is encouraged, and we have

taken advantage of this principle to include an introduction

iv
on the basics of meaningful measurement and the methodology

required to develop referee methods

v
PREFACE

Standard Reference Materials (SRM's) as defined by the

National Bureau of Standards are "well-characterized materials, produced in quantity, that calibrate a measurement

system to assure compatability of measurement in the nation.

SRM's are widely used as primary standards in many diverse

fields in science, industry, and technology, both within the

United States and throughout the world. In many industries

traceability of their quality control process to the national

measurement system is carried out through the mechanism and

use of SRM's. For many of the nation's scientists and technologists it is therefore of more than passing interest to know the details of the measurements made at NBS in arriving

at the certified values of the SRM's produced. An NBS series

of papers, of which this publication is a member, called the

NBS Special Publication - 260 Series is reserved for this

purpose

.

This 260 Series is dedicated to the dissemination of

information on all phases of the preparation, measurement,

and certification of NBS-SRM's. In general, much more detail will be found in these papers than is generally allowed,

or desirable, in scientific journal articles. This enables

the user to assess the validity and accuracy of the measure- ment processes employed, to judge the statistical analysis,

and to learn details of techniques and methods utilized for

work entailing the greatest care and accuracy. It is also

hoped that these papers will provide sufficient additional

information not found on the certificate so that new applications in diverse fields not foreseen at the time the SRM

was originally issued will be sought and found.

Inquiries concerning the technical content of this

paper should be directed to the author (s). Other questions

concerned with the availability, delivery, price, and so forth will receive prompt attention from:

Office of Standard Reference Materials

National Bureau of Standards

Washington, D.C. 20234

J. Paul Cali, Chief

Office of Standard Reference Materials

vi
OTHER NBS PUBLICATIONS IN THIS SERIES

NBS Spec. Publ. 260, Catalog of Standard Reference Materials,

i July 1970. 75 cents.* (Supersedes NBS Misc. Publ. 260,

[ January 1968 and NBS Misc. Publ. 241, March 1962.)

NBS Misc. Publ. 260-1, Standard Reference Materials:

j Preparation of NBS White Cast Iron Spectrochemical

! Standards, June 1964. 30 cents.*

INBS Misc. Publ. 260-2, Standard Reference Materials:

Preparation of NBS Copper-Base Spectrochemical

Standards, October 1964. 35 cents.*

NBS Misc. Publ. 260-3, Standard Reference Materials:

Metallographic Characterization of an NBS Spectrometric

Low-Alloy Steel Standard, October 196 4. 20 cents.*

(Out of print)

NBS Misc. Publ. 260-4, Standard Reference Materials:

Sources of Information on Standard Reference Materials,

February 1965. 20 cents.* (Out of print).

NBS Misc. Publ. 260-5, Standard Reference Materials:

Accuracy of Solution X-Ray Spectrometric Analysis of

Copper-Base Alloys, March 1965. 25 cents.* (Out of print).

NBS Misc. Publ. 260-6, Standard Reference Materials:

Methods for the Chemical Analysis of White Cast Iron Standards, July 1965. 45 cents.*

NBS Misc. Publ. 260-7, Standard Reference Materials:

Methods for the Chemical Analysis of NBS Copper-Base

Spectrochemical Standards, October 1965. 60 cents.*

NBS Misc. Publ. 260-8, Standard Reference Materials:

Analysis of Uranium Concentrates at the National

Bureau of Standards, December 1965. 60 cents.*

(Out of print)

NBS Misc. Publ. 260-9, Standard Reference Materials:

Half Lives of Materials Used in the Preparation of

Standard Reference Materials of Nineteen Radioactive

Nuclides Issued by the National Bureau of Standards,

November 1965. 15 cents.*

NBS Misc. Publ. 260-10, Standard Reference Materials:

Homogeneity Characterization on NBS Spectrometric

Standards II: Cartridge Brass and Low-Alloy Steel,

December 1965. 30 cents.*

vii
NBS Misc. Publ. 260-11, Standard Reference Materials:

Viscosity of a Standard Lead-Silica Glass, November

1966. 25 cents.*

NBS Misc. Publ. 260-12, Standard Reference Materials:

Homogeneity Characterization of NBS Spectrometric

Standards III: White Cast Iron and Stainless Steel

Powder Compact, September 1966. 20 cents.*

NBS Misc. Publ. 260-13, Standard Reference Materials:

Mossbauer Spectroscopy Standard for the Chemical Shift

of Iron Compounds, July 196 7. 40 cents.*

NBS Misc. Publ. 260-14, Standard Reference Materials:

Determination of Oxygen in Ferrous Materials —

SRM 1090, 1091, and 1092, September 1966. 30 cents.*

NBS Misc. Publ. 260-15, Standard Reference Materials:

Recommended Method of Use of Standard Light-Sensitive

Paper for Calibrating Carbon Arcs Used in Testing

Textiles for Colorf astness to Light, June 196 7. 20 cents.*

NBS Spec. Publ. 260-16, Standard Reference Materials:

Homogeneity Characterization of NBS Spectrometric

Standards IV: Preparation and Microprobe Characterizati

of W-2 0% Mo Alloy Fabricated by Powder Metallurgical

Methods, January 1969. 35 cents.*

NBS Spec. Publ. 260-17, Standard Reference Materials:

Boric Acid; Isotopic and Assay Standard Reference

Materials, February 19 70. 65 cents.*

NBS Spec. Publ. 260-18, Standard Reference Materials:

Calibration of NBS Secondary Standard Magnetic Tape

(Computer Amplitude Reference) Using the Reference

Tape Amplitude Measurement "Process A", November 1969.

50 cents.*

NBS Spec. Publ. 260-19, Standard Reference Materials:

Analysis of Interlaboratory Measurements on the

Vapor Pressure of Gold (Certification of Standard

Reference Material 745), January 1970. 30 cents.*

NBS Spec. Publ. 260-20, Standard Reference Materials:

Preparation and Analysis of Trace Element Glass

Standards. (In preparation)

NBS Spec. Publ. 260-21, Standard Reference Materials:

Analysis of Interlaboratory Measurements on the Vapor

Pressures of Cadmium and Silver, January 19 71. 35 cents viii
NBS Spec. Publ. 260-22, Standard Reference Materials:

Homogeneity Characterization of Fe-3Si Alloy,

February 1971. 35 cents.*

NBS Spec. Publ. 260-23, Standard Reference Materials:

Viscosity of a Standard Borosilicate Glass, December 1970.

25 cents.*

I|NBS Spec. Publ. 260-24, Standard Reference Materials:

Comparison of Redox Standards, January 19 72. $1.00.*

|NBS Spec. Publ. 260-25, Standard Reference Materials:

J A Standard Reference Material Containing Nominally Four

j Percent Austenite, February 19 71. 30 cents.*

NBS Spec. Publ. 260-26 , Standard Reference Materials:

National Bureau of Standards-U . S . Steel Corporation

Joint Program for Determining Oxygen and Nitrogen in

[ Steel, February 1971. 50 cents.*

NBS Spec. Publ. 260-2 7, Standard Reference Materials:

Uranium Isotopic Standard Reference Materials,

April 1971. $1.25.*

NBS Spec. Publ. 260-28, Standard Reference Materials:

Preparation and Evaluation of SRM's 481 and 482 Gold-Silve

and Gold-Copper Alloys for Microanalysis, August 19 71. $1.00.*

NBS Spec. Publ. 260-29, Standard Reference Materials:

Calibration of NBS Secondary Standard Magnetic Tape

(Computer Amplitude Reference) Using the Reference

Tape Amplitude Measurement "Process A-Model 2",

June 1971. 60 cents.*

NBS Spec. Publ. 260-30, Standard Reference Materials:

Standard Samples Issued in the USSR (A Translation

from the Russian), June 1971. $1.00.*

NBS Spec. Publ. 260-31, Standard Reference Materials:

Thermal Conductivity of Electrolytic Iron SRM 734 from

4 to 300K, November 1971. 35 cents.*

NBS Spec. Publ. 260-32, Standard Reference Materials:

The Cooperative Study of Temperature Scale Standards

for DTA by ICTA and NBS. (In preparation)

NBS Spec. Publ. 260-33, Standard Reference Materials:

Comparison of Original and Supplemental SRM 705,

Narrow Molecular Weight Distribution Polystyrene,

H. L. Wagner. (In preparation).

ix
NBS Spec. Publ. 260-34, Standard Reference Materials:

Thermoelectric Voltage. (In preparation).

NBS Spec. Publ. 260-35, Standard Reference Materials:

Thermal Conductivity of Austenitic Stainless Steel,

SRM 735 from 5 to 280K, April 1972. 35 cents.*

NBS Spec. Publ. 260-36, Standard Reference Materials:

A Referee Method for the Determination of Calcium in

Serum. SRM 736. (This publication)

NBS Spec. Publ. 260-37, Standard Reference Materials:

Methods of Analysis of NBS Clay Standards. (In

preparation)

.

NBS Spec. Publ. 260-38, Standard Reference Materials:

Preparation and Calibration of Standards of Spectral

Specular Reflectance. (In preparation).

NBS Spec. Publ. 260-39, Standard Reference Materials:

The Eddy Current Decay Method for Resistivity

Characterization of High-Purity Metals. (In

preparation)

.

*Send order with remittance to: Superintendent of Documents

U.S. Government Printing Office, Washington, D.C. 20 40 2. Remittance from foreign countries should include an additional one-fourth of the purchase price for postage.

x
CONTENTS

PAGE

I. INTRODUCTION 1 A. A Meaningful Measurement System ! B. Why Accuracy in Clinical Chemistry 4 C. The Role and Limitations of A Referee

Method (In General) 6 D. Background and History 7 II. THEORY OF OPERATION AND PRACTICE REQUIRED FOR

THE SUCCESSFUL DEVELOPMENT OF A REFEREE METHOD 9 A. Necessary Conditions 9 B. Modus Operandi U

III. HISTORY OF ACTUAL WORK 13

A. Definition of Terms Used ^3

B. Coding of Laboratories and Participation . . ^4

C. NBS Samples - Preparation or Source, Number

Coding, Calcium Concentrations 16

D. Analytical Procedures Used 19

E. Reporting of Data 20

IV. REFEREE METHOD - SCOPE, LIMITATIONS, PROCEDURE 21

A. Scope 21

B. Limitations 21

C. Procedure 25

V. COMMENTS OF THE COOPERATING LABORATORIES .... 35 Lab A 35 Lab B 35

Lab C 36 Lab D

37 Lab E

38

Lab F . . . 39 Lab H

40 Lab K

41 VI. RESULTS (JOHN MANDEL) 42 A. Introduction 42

B. Summary of Precision and Accuracy Data • • • 42

C. Discussion of Summary Table 44

D. Graphical Report of Results 45

xi
VII

.

THE ISOTOPE - DILUTION MASS SPECTROMETRY

WORK (LARRY J. MOORE)

A. Theory of Stable Isotope Dilution Analysis. . B. Mass Spectrometric Isotope Dilution as an Absolute Analytical Technique

C. Analytical Procedure

D. Analysis Results

VIII. DISCUSSION

A. Instruments

B. Reagents

C. Glassware

D. Aliquot and Dilution Steps

E. Sample

F. Instrument Reading and Valid Measurement

Criteria

G. Deviations From the Pybus et al . , Method. . . H. Discussion of General Points

IX. CONCLUSIONS

X. RECOMMENDATIONS FOR FUTURE REFEREE METHOD

DEVELOPMENT STUDIES

A. Preplanning Session

B. Coordinator

C. Financial Support

D. Experts Committee

REFERENCES . APPENDIXES

xii
LIST OF TABLES

PAGE

Table No .

1. Laboratory Code Letters and Exercise

Participation 15

2. NBS Samples: Composition, Source, Lot and

Sample Code Numbers, and ID-MS Values .... 18

3. Instruments Used and Operating Conditions . . 2 3 4. Flame Conditions, Pressure and Flow Rates . . 2 4 5. Summary of Precision and Accuracy Results . . 4 7 6. Concentration of Calcium in Synthetic CaCO-. Solutions, meq/liter. (Exercise 1) 7 3 7. Concentration of Calcium in Synthetic CaCO^

Solutions (Na and K added) , meq/liter

(Exercise 2) 76

8. Concentration of Calcium in Serum, meq/liter

(Exercise 3) 78

9. Concentration of Calcium in Serum meq/liter

(Exercise 4) 79

10. Concentration of Calcium in Serum, meq/liter

(Exercise 5) 82

xiii
LIST OF FIGURES

PAGE

Figure No.

1. Typical Calibration Curve for the atomic Absorption Determination of Calcium 32

2. Percent Deviation from ID -MS Target Values

.

48

3. Percent Deviation from ID -MS Target Values

.

49

4. Percent Deviation from ID -MS Target Values

.

50

5. Percent Deviation from ID -MS Target Values

.

51

6. Percent Deviation from ID -MS Target Values

.

1 52

7. Percent Deviation from ID-MS Target Values.

Laboratory A 53

8. Percent Deviation from ID-MS Target Values.

Laboratory B 54

9 . Percent Deviation from ID-MS Target Values

.

Laboratory C 55

10. Percent Deviation from ID-MS Target Values.

Laboratory D 56

11. Percent Deviation from ID-MS Target Values.

Laboratory E 57 12. Percent Deviation from ID-MS Target Values.

Laboratory F 58 13. Percent Deviation from ID-MS Target Values.

Laboratory H

5 g 14. Percent Deviation from ID-MS Target Values.

Laboratory K 60 15. Percent Deviation from SRM 915 (CaC0

3

) Weighed in Value 61

16. Percent Deviation from Group Average 6 2 xiv
A REFEREE METHOD FOR THE DETERMINATION OF CALCIUM IN SERUM

J. Paul Cali, John Mandel, and

Larry Moore

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

and

Donald S. Young

National Institutes of Health

Department of Health, Education

and Welfare

Washington, D.C. 20214

The first referee method of analysis (a method

of known accuracy) in clinical chemistry has been

developed by a team of experts from clinical chemistry, 8 cooperating clinical chemistry laboratories,

and scientists from the National Bureau of Standards.

Subject to the limitations and provisos stated, calcium in serum can be determined by the referee method to within +2% of the "true or absolute" value. Al

so discussed are: (1) What Constitutes a Meaningful

Measurement System; (2) Theory of Operation and

Practice Required to Develop a Referee Method; (3) History of the Actual Work; the Referee Method Protocol; (4) Results; (5) The Isotope-Dilution

Mass Spectrometry Work; (6) Discussion, Conclusions

and Recommendations for Future Referee Method Develop

ments

.

Key words: Accuracy; atomic absorption spectrometry;

calcium in serum; clinical chemistry; referee method

for calcium.

xv

! I. INTRODUCTION

A. A Meaningful Measurement System

In 196 6, Vannevar Bush said, "If men are to accomplish

i together anything useful whatever, they must, above all, be

J able to understand one another." Understanding between men

I implies communication, and in science and technology, effecj tive communication between and among scientists, engineers,

j technicians, and all those involved in the various aspects

j of scientific and technological endeavors, is realized, in

: large part, through the measurement process. However, the

degree to which the communication is useful and effective

depends largely on whether the measurements made are in

actuality meaningful . When a measurement is meaningful,

the value or number associated with the property under

I consideration meets three criteria—the number obtained

' during measurement truly reflects in some well-defined way

the property being measured, and the number is both accurate

and precise. In ideal cases, and certainly in "pure" measureI ment science, a direct relationship can be traced, usually

quite straightforwardly between the property measured and

the base units of the Systeme International. The realization

of the candela, insofar as the material aspects of the measurement are concerned depends only on the purity of the platinum

since the candela is defined in terms of that material. In this instance, meaningful measurement (or realization) of the

candela depends upon knowing the purity of the platinum to be

used to a high degree of accuracy and precision.

As we move down the ladder from the realm of "pure"

measurement science into the murkiness of applied measurement, the task of making meaningful measurement becomes

ever increasingly difficult. In the United States in 19 71

9 more than 10 measurements were made in over 20,000 clinical

laboratories. Yet if the three criteria mentioned above

1
are applied to these measurements, then it is certain that

a large fraction, perhaps more than 25%, are not meaningful

at all. It is instructive to examine in some detail what constitutes a meaningful measurement system . Too often scientists

and engineers concentrate on one aspect of a system, not

realizing that unless all parts of the system are tended and

cared for, that the hoped-for-result will not be achieved.

A universal, meaningful measurement system consists of

five distinct parts:

1. A rational, self -consistent system of units of measurement. (E.g., the S.I., now universally accepted.)

2. The means to realize in practice the defined units

and their derivatives, for example, through the use

of certified standard reference materials. In areas

of applied technology the production and availability

of well-characterized materials is of utmost importance in allowing the transfer of meaningful measure- ment by these means from highly competent laboratories

that measure and produce these materials, to the user

in the applied sector.

3. Methods of measurement or analysis or test, based

upon the well-characterized materials of part 2, and

which are, above all, accurate methods. Only in a system based on accuracy can communication between

different fields of science and technology be effective. Precision alone is insufficient. These basic,

accurate methods are often referred to as referee

methods , reference methods , or standard methods . In clinical chemistry the first term, referee methods,

is the phrase of choice. In any case, they should

be methods of proven accuracy. They need not be, nor are they often in practice, the methods applied

or used in industrial or technological application.

2
They, together with the well-characterized materials

of part 2, are available to test and check the methods

of part 4, below.

4. Field or applied methods of measurement or analysis

or test. These are the methods applied on a large

scale in everyday work. They have been introduced,

often at the expense of accuracy, to turn out a large volume of results. However, if the methods

of part 3 are available, then there is no fundamental reason why these practical methods should

not be checked or validated against the referee

method to assure their accuracy. Of course, if the

referee method of part 3 can be made to meet the

requirements of the field methods (speed, ease of

operation, low-cost, etc.) then the referee method

and field method should be made identical.

5. A method whereby the long-term integrity of the measurement system is assured. Given that parts 1-4 are operative and under control, then a mechanism

is set up whereby the various practitioners and users

of the system are periodically tested against materials supplied by a checking or validating agency

(industrial, governmental, professional society,

etc.). The properties of the material to be tested

are known only to the checking agency via parts 2 and 3. Returned results are statistically analyzed

and it is easily determined whether the measurement

process in individual laboratories or an entire

industry is in fact under control.

Obviously, the implementation and operation of a meaningful measurement system for all science, industry and

technology is far beyond the ability and resources of any one organization. The work required is so vast in scope

that only through a large network of qualified and inter3
ested organizations can order be brought to bear. In the

steps outlined above, the organizations listed below should

bear some responsibility for implementing and/or continuing

their efforts to provide the required resources to bring

about meaningful measurement

:

Part 1 - International Committee of Weights and Measures

(CIPM) ; National Standards Laboratories; International

Union of Pure and Applied Chemistry (IUPAC)

.

Part 2 - National Standards Laboratories; Qualified Manufacturers; IUPAC and CIPM (information dissemination);

Professional Societies.

Part 3 - Professional Societies; Standards Bodies (e.g.,

ISO, ASTM) ; Governmental Agencies and Laboratories.

Part 4 - Commercial Laboratories and Associations;

Standards Bodies; Professional Societies.

Part 5 - Governmental Agencies and Laboratories; Commercial Laboratories and Associations; Standards

Bodies

.

B . Why Accuracy in Clinical Chemistry

In the preceding section, stress was laid on accuracy in

measurement. Some confusion still exists as to the exact

meaning of accuracy, especially as opposed to precision.

One of the best discussions on this subject was given by

C. Eisenhart [1]. A paper titled "The Attainment of Accuracy in Clinical Analytical Methods" applies more particularly to the field of clinical chemistry [2] . When the value

obtained by the measurement process for the property under

consideration is essentially the same as that obtained by

any other acceptable measurement process, or, alternatively,

is the value predicted by a well-developed theory, then

accuracy has been achieved. The key word in the above is

"acceptable". An acceptable measurement process is one the

accuracy of which has been established by some other independent route, and its remaining systematic biases (never

4
totally reduced or even known with certainty) are small

relative to the accuracy goal of the measurement process

under test. Clearly, in practice, there will be many instances where neither an acceptable measurement process is

available nor where theory can be relied on to assure accuracy. In such cases, consensus, by experts, is probably

the only viable alternative. Such a route, however, should

be considered a stop-gap measure and additional work performed to establish an independent method of known accuracy.

The question often arises among scientists in clinical

chemistry, "Why is accuracy needed, in any case, as in

practice^ very large uncertainties in the overall, total

medical picture remain?" There are several answers to this

question. First, there is a legal argument. No matter how

precise a method is shown to be , the values obtained by a precise but inaccurate method cannot and will not be sus- tained under legal challenge if values obtained by a method

shown to be more accurate are offered in evidence. A more

accurate value is by definition "closer to the truth," and

would tend to carry more weight in legal disputes

.

Second, and more important from the scientific viewpoint,

is a transfer of information argument. Given two inaccurate

but precise measurement processes, both capable of measuring

the same property and each giving different values for that

property, it is difficult to determine which of the values

should be accepted. Statistical analysis has little to say

about systematic biases and it cannot be ascertained statistically whether one or the other or both of the methods

are inaccurate, the direction of the biases, the magnitude

of the corrections, etc., because -there is no base-line

reference against which these parameters may be measured.

Even greater difficulties are encountered when measurements

on different properties are to be evaluated and correlated

as, for example, the determination of the mass balance of
a system. Only when the measurement processes give accu- rate values are these difficulties overcome.

Hence, the goal of every measurement system should be one of accuracy. Further advances should not be limited

by inability to measure accurately but rather by limitations

in the fundamental knowledge of the system under study.

C . The Role and Limitations of a Referee Method (In General)

As was explained in section I-A, above, a referee method

is a measurement method of known and proven accuracy, i.e.,

the systematic errors or biases of the measurement process

have been found, mechanisms elucidated, and these errors eliminated, or, as~ is sometimes the case when actual physical

elimination is impossible, corrected or accounted for. There

is some misunderstanding as to the limitations and scope of

a referee method:

1. A referee method is not, per se , to be a legally

imposed method, the preferred method, a method to be

used for qualification or certification of laboratories and the like. It may, in fact, become any

of these if various organizations, assumed to have

the power to so declare by law, fiat, etc., do so choose to have the referee method serve these purposes . 2. A referee method is not necessarily an easily used

method; it does not have to be a method which can be

used by the average practitioner in any given field.

It may in fact require special skills, special equipment, controlled environment, etc., which are not

readily or easily available to the average laboratory.

It should not, however, be so highly specialized or unique that only a handful of scientists can use the

method. This latter criterion depends to some extent

on the particular field of science. In metrology

there are only a small number of national laboratories
who can realize the measurement of time to an accu12 racy of 1 part in 10 , but because this measurement

at a high accuracy is readily transferred when needed

elsewhere, not many principal practitioners are re- quired. In clinical chemistry, the situation is much

more complex. Here the referee method should be realizable, in the real world, by many dozens of qualified

laboratories so that transfer of the proven accuracy

from the referee method to the dozens of field methods

may be accomplished in some reasonable time span at

a reasonable expenditure of time, energy, and funds.

In sum, a referee method is a method of known accuracy,

useable in the real world of measurement, one necessary link

in a chain of events that leads to a meaningful measurement

system.

D . Background and History

In 1967 the National Bureau of Standards (NBS) acting on

the recommendations of the Standards Committees of the American Association of Clinical Chemists (AACC) , the College of

American Pathologists (CAP) , and with the subsequent support

of the National Committee of Clinical Laboratory Standards

(NCCLS) initiated a program, largely with financial support

from the National Institute of General Medical Sciences

(NIGMS) , to produce, certify, and issue Standard Reference

Materials (SRM's) for use in clinical chemistry. This program has resulted to date in the issuance by NBS of 14 clinical SRM's [3], Until 1969, NBS did not consider ex- panding further afield in the total clinical measurement

system. However, at the International Federation of Clinical Chemistry meeting in Geneva, Switzerland in September

1969, NBS was urged by the Executive Board of that body to consider playing a coordinating role in the development of

referee methods that were seen to be urgently needed.

Dr. George Bowers, Jr., Hartford Hospital, and Dr.

7
Donald Young, National Institutes of Health, during the first

half of 1970 were instrumental in helping shape the thinking

and planning at NBS for the initiation of a plan whereby

referee methods for clinical chemistry could be developed,

tested, and validated.

Several of the subsequent participants in this work met

at Hartford Hospital in May 1970 and later at NBS in June

1970 where agreement in principle was reached on the main

tasks and routes to be pursued. Valuable critical advice

at this stage was provided by Drs . Alan Mather and Nathan

Radin, Center for Disease Control (CDC) and Dr. Roy Rand,

Hospital of the University of Pennsylvania. As a result

of these discussions, J. Paul Cali , NBS,, prepared a memo- randum that laid down, from the NBS point of view, the minimum

requirements and ingredients necessary for the successful

development of a referee method (Appendix 1)

.

The actual work began on September 10, 1970, at a meeting at NBS, when the accuracy goal for calcium in serum

was set, the candidate method chosen, and various other

details agreed on. The achievement of a referee method for the determination of calcium in serum was accomplished in December 1971.

8
II. THEORY OF OPERATION AND PRACTICE REQUIRED FOR THE SUCCESSFUL DEVELOPMENT OF A REFEREE METHOD

A. Necessary Conditions

For the successful attainment of any Referee Method the

following conditions must be met or defined:

1. An authoritative Standard Reference Material (SRM)

must be available. It should be a highly homogeneous substance, usually of the highest possible

purity, and its properties should be certified by

a laboratory or group of the highest technical

competence. (E.g., SRM's issued by National

certifying bodies)

2. A method of analysis having high precision must be

selected for study as the candidate for development

as the Referee Method. Considerations of ease of

operation, cost, and time are secondary to the

achievement of a method having a known accuracy.

3. There should be a clearly defined accuracy goal

for the Referee Method.

4. Laboratories of recognized technical competence

must participate in the interlaboratory work.

5. The system must be in a state of quality control

at all times. Quality control should be assured

and maintained by properly designed and executed

statistical methods under qualified and experienced

statisticians

.

6. A panel of qualified experts must review and direct

all the technical phases of the work. As a pragmatic

consideration, it is best that the coordination of

the entire operation be focused in one responsible

person

.

7. If possible, an independent method having a known

accuracy of at least a factor of 2 better than the

9
stated accuracy goal of the Referee Method under

test should be used to measure the property (ies

)

under consideration, for the test samples sent to the cooperating laboratories. The question immediately occurs: If an independent, accurate

method is available, why then cannot it be declared the referee method? In many instances, this

does not happen because the independent method is too complex and involves specialized equipment and

skills which are not readily available in the field

under study. As will be made evident this situation

applies in this study. (See more detailed explanation

in section VIII-H.

)

In the calcium Referee Method developed and reported

here the above conditions were met as follows

:

Under Al : NBS-SRM Calcium Carbonate (SRM 915) was available and used throughout all five exercises.

The certificate of analysis for this SRM is shown

as Appendix 2

.

Under A2 : The Experts Committee chose as the candidate

method an atomic absorption method for calcium based

on the work of Pybus , Feldman, and Bowers [4], Re- visions and modifications to improve accuracy were made during the course of the work and are incorporated in the final referee method that is given

in full detail in section IV. under A3: The Experts Committee set two accuracy goals

with the expectation that the more stringent, while

desirable, was not likely to be attained. These were a method giving an accuracy to within ±0.5%

(desirable) and ±1.0% (probable) of the true value.

As work progressed, tacitly it was agreed that the

latter limit was in fact the goal for this method.

Under A4 : The Experts Committee chose eight cooperating

laboratories to participate in the interlaboratory
work

.

The names of the principal investigator and the complete

addresses are given in appendix 3. Under A5 : Statistical advice, guidance, and evaluation

were performed by Dr. John Mandel, Staff Statistician,

Institute for Materials Research, NBS

.

Under A6 : The Experts Committee originally consisted of Drs . G. Bowers, N. Radin , and D. Young. Advice and

criticism during the course of the work was made by

Dr. J. Boutwell (CDC) and Dr. R. Eilers (CAP).

Under A7 : Fortunately, at NBS an independent method

for the accurate determination of calcium using

an isotopic dilution mass spectrometry technique

was available and used fruitfully in this work.

This method served as the independent base line

and is fully described in section VII, below.

This part of the work was under the overall

direction of William R. Shields.

B . Modus Operandi

All the necessary conditions were met and fulfilled by

September 1970. The work and interrelationships of the

Experts Committee (A6 , above), the cooperating laboratories

(A4 , above), and the statistical team (A5) was coordinated

at NBS through the Office of Standard Reference Materials

(OSRM) under the guidance of J. Paul Cali , Chief of that

office

.

The actual steps followed in the work were these:

(See note following)

Step 1 - Define the goals of the Referee Method in terms

of what is to be achieved, use of the method, applicability, limitations, etc. (EC)

Step 2 - Set accuracy goal. (EC)

Step 3 - Provide an SRM whose purity, or other certified

property, is known more accurately than the accuracy

11
goal set for the Referee Method. (NBS)

Step 4 - Choose an appropriate clinical analytical method

as a candidate for the Referee Method. (EC)

Step 5 - Set forth the experimental and statistical

designs. (EC + NBS)

Step 6 - Provide the SRM in solution and serum (or

another matrix, as required) at several concen- trations that are known to an accuracy greater

than the accuracy goal of the Referee Method. (NBS)

Step 7 - Provide detailed work procedures for cooperating laboratories. (EC + NBS)

Step 8 - Perform laboratory experimental work using

SRM solutions (for calibration purposes) and then

analyze unknowns (both solutions and sera) distributed by NBS (concentrations known only by NBS). (CL)

Step 9 - Analyze results from cooperating laboratories.

Elucidate and evaluate suspected biases in the can- didate method. Modify procedures to eliminate sus- pected sources of error and to control variables

within tighter limits as required. (EC + NBS)

Step 10 - Repeat steps 6 through 9 until accuracy goal

is achieved. (EC + NBS + CL)

Step 11 - Publish analytical method as a Referee Method.

(EC + NBS)

(Note: Groups responsible for the various steps are shown

in parenthesis after each step. The following abbreviations

are used: Experts Committee (EC); coordination, statistical

services, SRM and sample distribution (NBS); Cooperating

Clinical Laboratories (CL)

.

12
III. HISTORY OF ACTUAL WORK

A. Definitions of Terms Used

1. Exercise : The work performed by the cooperating

laboratories on a specific lot of either

calcium solutions or pooled sera containing calcium.

Included are: (a) the instructions for that particular exercise, (b) the actual samples, (c) the

data produced and analyzed, and (d) the work done at NBS on the same lot to produce values of the calcium

concentration by the isotope dilution-mass spectrometric method.

Five different exercises were required. From initiation

to finish, each exercise averaged two months. The various

exercises are numbered from 1-5, serially.

2. Run : A complete set of measurements made by a particular laboratory on a specific, complete lot

of samples in a single session of work.

In each of the five exercises, each individual sample

was run in duplicate, but with the order for the entire set specified in advance. Two runs, in each of two successive

weeks, were required for each set of samples for each

exercise. (Exceptions will be noted in the tabular data)

3. NBS Samples : Vials or bottles containing either

water solutions, calcium, or sera, and individually

numbered. Calcium concentrations, determined by

the isotope dilution-mass spectrometric (ID-MS)

method, were not known in advance nor given to the

cooperating laboratories during the course of any

exercise. (One exception noted in tabular data)

4. Glassware, water, reagents, etc. : These, together

with their specifications, are defined in the protocol, section IV.

13
B. Coding of Laboratories and Participation

Laboratories were assigned a letter. Not all laboratories participated in every exercise for reasons that

are explained below. Letters assigned and exercises participated in are shown in Table 1. Explanation for incomplete data or abort or for non- participation are as follows:

1. Lab D - Exercise 2: samples held up in shipment;

several weeks storage in warm area; finally, instrument breakdown. No data used in Exercise 2. 2. Lab E - Exercise 4: Instrument instability, could

not meet precision requirements; also, condition of

sample led investigator to believe not able to perform quantitative transfers. No data used in Exercise 4.

3. Lab F - Exercises 4 and 5: After protocol tightened

up (after Exercise 3) , instrument used could not

meet precision requirements. No data available

for Exercises 4 or 5.

4. Lab K - Exercises 1 and 2: No participation until

Exercise 3. No data available for Exercises 1 and

2.

14
Pn Q Q Q ft ft u u u z a H Q Q Q Q Q U U U H U

^0ou

>1o

+J

tauoa

-P U

to <d -h P Q -P

IT) M Q (D <tf -P ft

Q) O +IH+)

<D & O H£2 a o 5 c-h U H Q I I I Q Q ft U H 2

15
C . NBS Samples —Preparation or Source, Number Coding

,

Calci~um Concentrations (By ID-MS)

Exercise 1 - Three solutions of calcium at concentrations

of (approximately) 0.08, 0.10, and 0.12 g of Ca/1 were prepared at NBS using SRM 915, Calcium Carbonate.

Solvents were isothermally distilled HC1 and redistilled water.

Exercise 2 - Three solutions of calcium at concentrations

of (approximately) 0.07, 0.10, and 0.13 g of Ca/1 were prepared at NBS using SRM 915, Calcium Carbonate, in

which 3.2 g of Na/1 and 0.2 g of K/l were added using

SRM 919, Sodium Chloride, and SRM 918, Potassium

Chloride, respectively. The same solvents, as in

Exercise 1, were used.

Exercise 3 - Four, one liter bottles (polyethylene) of

pooled patient serum were received from Hartford

Hospital. The four lots, their ID numbers (at

Hartford) and approximate calcium concentrations were: Lot 1 - serum pool mixed 26E and 27E, prepared

August 1970, Ca ^6.0 meq/1

.

Lot 2 - serum pool 27A, prepared December 1970,

Ca ^4.5 meq/1.

Lot 3 - serum pool mixed 26 (B + C) , prepared

August 1970, Ca ^5.0 meq/1.

Lot 4 - serum pool 26A, prepared August 1970,

Ca ^4.8 meq/1.

Exercise 4 - Same as Exercise 3, except that proportions were changed to give slightly different calcium

concentrations as follows

:

Lot 1 - ^5.1 meq/1

Lot 2 - ^5.8 meq/1

Lot 3 - ^3.6 meq/1

Lot 4 - M.l meq/1

16
The sera used in Exercises 3 and 4 were prepared at Hartford Hospital according to procedures given in reference [5].

For reasons which are not entirely clear, these sera

samples, after repackaging in flame -sea led vials at NBS

,

started to show mold and bacterial growth. It is suspected

that NBS 1 inexperience with sterile techniques and the fairly

long time between shipment from Hartford to NBS and then to

the cooperating laboratories were all contributing factors.

The deterioration of the serum used in Exercise 4 was, however, not evident until after repackaging and was only apparent

several days after shipment to all the participants. The NBS

samples for these two exercises were far from ideal and much

worse than sera samples collected under usual field conditions.

In addition to the four lots of sera sent out in this

exercise, an aqueous acid solution of the NBS-SRM CaCO^ (SRM

915) was again prepared and sent with the four sera samples.

The calcium value was ^5.5 meq/1. It also contained sodium

and potassium at the same level indicated in Exercise 2. Exercise 5 - The sera for this exercise was prepared

at CDC, Atlanta, Georgia under the supervision of

Dr. David Bayse. Twenty vials of each of four

concentrations and containing 50 ml of bovine

pooled sera were received at NBS

.

Lot 1 - -V3.6 meq/1

Lot 2 - ^4.3 meq/1

Lot 3 - ^5.0 meq/1

Lot 4 - ^5.7 meq/1

The exact procedure used in the preparation of Exercise

5 sera samples is given in Appendix 4

.

All the data of this section plus the actual sample

number codes and the ID-MS values are summarized in Table 2.

17
-PO W3

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18
D. Analytical Procedures Used

The candidate referee method under development is

based on the method for determining calcium in serum by

atomic absorption spectrometry developed by Pybus , et al [4].

When a general method is adapted for a particular use, under specified conditions, then that particular write-up

is called a procedure, or as widely used in clinical chemistry, a protocol.

Three procedures based on the Pybus, et al, method were

used in the course of this work:

1. In Exercise 1, each laboratory was permitted to use the Pybus method (as if it were a detailed procedure)

as published in Clinical Chemistry. Specific details

not expressly stated were left to the discretion of

each participant. Significant departures were to be reported. Calibration of the atomic absorption

spectrometer was to be by an accepted procedure using

a calibration curve and the procedure used was also

to be reported. Finally, any deviation from reagent

specifications was to be reported.

2. In Exercises 2 and 3, the Pybus method was amended

to provide more specific instructions in several

areas. In addition, clarification of several points

was made. The first revised procedure will not be

given here. The revisions included: (a) a glossary

of terms used, which had caused some confusion in

Exercise 1, (b) a brief explanation of how the ID-MS

method would be used to provide the reference base-line

against which the interlaboratory results would be

measured, (c) more detailed instructions on the

preparation of the various solutions used, (d) a clarification of the section specifying the order in

which the blank, standard solutions and unknowns are

to be determined, and (e) a modified reporting procedure so that all ten readings of the absorbance
for a given unknown would be recorded.

3. In Exercises 4 and 5, a further revision was made,

following a meeting at NBS of all participants

,

to tighten up several steps. These included:

(a) more rigid pipetting instructions, including

a technique to insure good rinsing while saving

sample solution, (b) an exposition of what con- stituted a valid measurement and explicit rules

for discarding data, (c) an exposition of the

procedure to be followed in the determination,

(d) reporting of significant figure, (e) importance

of temperature control, (f) re-emphasis on quality

of water used and specifications for reagents , and

(g) emphasis on assuring the stability of the

spectrometer

.

4. The final procedure, rewritten for this publication,

is given in section IV.

E. Reporting of Data

Each cooperating laboratory received with the actual

samples, data sheets for recording all pertinent data.

The forms used are shown in Appendix 5. In addition,

comments regarding deviations from the protocol, troubles

encountered, suggestions for improvement, etc., were returned to NBS with the completed data sheets for each

exercise

.

20
IV. REFEREE METHOD - SCOPE, LIMITATIONS AND PROCEDURE

j A. Scope

The procedure given in Section C, below, is derived and

I adapted from the candidate method of Pybus , et al [4].

j The referee method when used exactly as given, subject to

| the limitations stated, will give the concentration of calcium

| in serum with an accuracy within ±2% of the true value.

The referee method as given here is not to be considered

| suitable for routine use in the clinical laboratory. The

| size of sample required, precautions that must be adhered

j to, time required, and cost preclude its use under normal

operating conditions. This should not be construed to mean

that this method cannot be adapted for routine use as indeed

was the case in the original Pybus, et al., method. Its primary

utility lies in the following:

1. To establish the absolute value of calcium in control

or pooled sera.

2. To establish the validity of calcium values given

in various clinical kits.

3. To be a method against which the many field methods

for calcium may be tested to ascertain the accuracy

of those methods

.

4. To be used by manufacturers of calcium reference

materials, kits, control sera, etc., in establishing

and maintaining the quality of these products . B. Limitations

During the course of these studies , several important

limitations became evident. The following conditions must

be met to attain the accuracy inherent in the referee

method

:

1. The procedure must be adhered to in all details.

Deviations, short-cuts, adaptations, etc., are not

allowed.

2. All glassware involved in volumetric measurements,

21
i.e., pipets and volumetric flasks, must meet NBS

Class A specifications. These items are commercially

available

.

3. Reagents, including water, must meet specifications

set forth in the procedure.

4. In this work three different spectrometers were used

(see Table 3). All are double-beam instruments.

It is not possible to say whether or not other instruments can provide the necessary stability, precision,

etc. , to accomplish the obtainable accuracy of the

referee method. In one instance, Lab F dropped out

of Exercises 4 and 5 when it was determined that its

instrument could not meet the precision requirements

set forth. It is clear that the instrument used must

be in optimum operating condition, highly stable, and

linear throughout the range of interest. Flame

conditions used are shown in Table 4.

5. The work must be performed by workers well acquainted

with the techniques of analytical chemistry. Weighings, aliquoting, preparation of standard, blank,

and unknown solutions must be performed with the

highest regard to accuracy in order to limit errors from these steps to much less than 1%, if the overall accuracy of the method is to be achieved.

6. Sufficient, uninterrupted time (^1 week) must be made

available to perform the method. It cannot be worked

into the daily work load of the clinical laboratory.

Finally, the worker who performs the method must be

highly motivated. This implies a scientist who enjoys

working meticulously and wants to provide analytical

data that will stand up to the most critical analysis.

22
CD u P 0 +> U u U u m u u a P o 0 o 0 o o

(0 Cn u VD in r» r» cn u> H er an om

CN1 CN CN CN1 CN1 CN CN1 Cu OS CN O oo rH CN 00 ro rH

em CN CN CN CN

in

CN CN

Eh CN

rH

(0 •H

-P Q 0 P Cn

r> rH rH CO rH C rH P

cd re rd rd T3 m •H rd cd CO cu -p 4-> +J P p -P -p +J c

ps •H H 0 H rd •H p

tr Cn O tr> P Cn Cn 13 -H •H CO -H O pq Q Q Q OS Q PS b Q

CJ u o c

•H c p Cn p P rH 0 p C p P •H

•H CO -H CO CO 0 p o Cn E S GQ rl 8 Cn C Cn Cn 0 0

•rl C •H C C H fd c •H CO N •H H Xi 0 o O p O O o u w u -H T3 •H •rl rd o Tl Tl 13 o O p CO rH CO CO p P

(0 P to O P P w to 10p

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CN CN CN CN CN CO CO CN rd rH CJ u U u CJ > > U P

CO rl •H 0 1 Cn Cn

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•rl •H •rl -rl •rl O O -P < < a C < rH

rH

00 00 ro ro CO o oo o o o O ro 00 CO 1

in

rH

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rH

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rH ta l3 PI P H 0< P-l Pn H H 3

23
Table 4. Flame Conditions

,

Pressure and Flow Rates

Air Acetylene

Lab

Code PSI 1/min PSI 1/min

A 30 21.2 10 8-9 B 37-38 8.8 5.4-6 1.7

C 30 16^ 8-8.5 4 D 30 7.5 8-8.5 9 E 8.5 NR 9 NR

F 28 NR 8.5 NR

H 14 NR 4 NR

K 5 NR 5.5 NR

NR - Not Reported

a/ - Exercise 2 only 7.5 1/min

24
Procedure

Samples

The method is designed to provide accurate values of

the calcium concentration of serum or aqueous solutions

as provided for analysis. The method does not assure adequacy or integrity of sampling per se, but if a re- presentative sample is taken, the method will provide an accurate value of the calcium in that specimen. Whether

the specimen is truly representative, or homogeneous,

is beyond the scope of this method. The only requirement is that a representative sample of the presented

specimen be pipetted accurately. The method has applicability, at the specified accuracy, in the range from

3 to 7 meq Ca/1.

Reagent Specifications

1. Water, preferably distilled and deionized, should measure at least 10^ ohms specific resistance at

25 °C. It should be available in large quantity

for use as a diluent and for the final rinse operation on all glassware and apparatus coming in

contact with the solutions involved. Only water

that meets these specifications is to be used in

these operations

.

2. Calcium standard solutions should be prepared from

CaCO^ issued and certified by NBS . Its identification number is SRM 915. This material should be

dried for 4 hours at 200 °C and cooled to room temperature in a desiccator before use.

3. Lanthanum oxide should be of high purity and known

to contain less than vL5 yg of calcium per gram.

4. Sodium, potassium, and strontium chlorides should

be ACS Analytical Reagent Grade (AR) quality.

5. Hydrochloric acid, meeting ACS-AR specifications

should be used.
Glassware Specifications

All glassware required—10 ml volumetric pipet (to

contain) ; 500 ml volumetric flasks —should meet NBS

Class A specifications.

All glass or plastic surfaces coming into contact

with reagents, water, diluent, or sample must have

been previously cleaned as follows:

1. Use routine cleaning procedure (hot water

with detergents, plus usual rinses)

2. Soak glassware for 30 minutes in 0.05 M HC1.

3. Rinse with several portions of distilled water,

(5-6 minimum)

4. Air dry (inverted) in a dust-free environment.

Preparation of Reagents

1. Stock Blank Solution (140 mmol in NaCl and 5.0 mmol

in KC1 per liter) . To a clean 1-1 volumetric flask,

add 8.18 g of NaCl and 373 mg of KC1. Dissolve in

H^O and fill to the neck. When at working temperature

(ambient) , dilute to calibrated volume and mix by inverting the flask 30 times.

2. Diluent Solution (10 mmol LaCl^ and 50 mmol HC1 per

liter) . (Plan to make sufficient diluent for the work

to be performed in one continuous series.) Transfer

1.63 g of La

2 0^ to a 1-1 flask and dissolve in 10 ml

H

2

0 and 6.7 ml concentrated HC1. After the La

2

0 3 is dissolved, dilute with water to the neck of the

flask. When the solution has reached ambient temperature, dilute to calibrated volume and mix by inverting

the flask 30 times. (NOTE: If an internal reference

is to be used, add 30.6 mg SrCl

2 *6H

2

0 per liter)

3. Standard Stock Solutions of Calcium. Prepare a minimum of three concentrations at 4.00, 5.00,

and 6.00 meq Ca/1 with each to contain 140 mmol NaCl

26
and 5.0 mmol KC1 per liter. To each of three 1-liter

volumetric flasks, add 8.18 g NaCl and 373 mg KC1. To the first flask (4.00 meq Ca/1) add 200.2 mg of

CaCO- to the second flask (5.00 meq Ca/1) add 250.2

mg CaC0 3

, and to the third flask (6.00 meq Ca/1) add

300.2 mg CaC0

3

. (Use only NBS-SRM 915 previously dried

at 200 °C.) To each flask add a few ml of H

2

0 and 1 ml concentrated HC1. Make sure all the CaCO^ is in

solution before diluting with E^0 to the neck. When

at ambient temperature, dilute each flask to calibrated

volume and mix by inverting the flask 30 times.

Label all flasks appropriately.

Procedure for Diluting

All solutions should be at ambient temperature.

All solutions, except the diluent, but including the

unknown samples, will be diluted to 1:50 using a 10 ml

volumetric (TC) pipet and 500 ml volumetric flasks. Only

one 10 ml volumetric pipet is to be used throughout to reduce errors caused by differences in drainage times

between the aqueous or dilute acid and sera solutions.

1. Transfer to a 500 ml volumetric flask approximately 450 ml of diluent stock solution. Add

to the flask 10.00 ml of the blank stock solution using the 10 ml pipet. (NOTE : The tip

of the pipet should remain in the diluent to prevent the formation of foam by the serum)

After drainage of the pipet stops, using

a rubber bulb, gently blow out the residual

liquid. Rinse the pipet three times with diluent

from the flask, each time returning the pipet

contents to the flask by drainage and blowing.

2. Dilute to calibrated volume with diluent and

mix thoroughly with 30 inversions. Set aside.

3. Draw into the pipet H„0 contained in a clean

27 A
beaker. Fill to slightly above the mark and

discard

.

To condition the pipet, fill to 1-2 mm above

the mark with 4.00 meq Ca/1 standard stock solution. Discard. Repeat twice. To a 500 ml volumetric flask, transfer 10.00 ml

of the 4.0 0 meq Ca/1 standard stock solution

using the technique described in steps 1, 2, and

3 , above

.

Repeat step 5, twice, but using the 5.00 and

6.00 meq Ca/1 standard stock solutions. Condition

the pipet each time as given in step 4 , using the

approximate standard stock solutions

.

After the blank and standard solutions have been

diluted and the pipet rinsed with f^O, draw 2-3 ml

of the first unknown solution into the pipet.

Place a finger over the end of the pipet and

withdraw from unknown solution container. Tilt

the pipet to a horizontal position and slowly

rotate the pipet to wet thoroughly all internal

surfaces. Allow a small amount of air to leak

past the finger so that the rinse solution may

come into contact (a small way above the mark)

with the upper stem surface. Discard. Repeat

the rinse and conditioning operation once more

.

Fill the pipet to the mark with the unknown

solution and deliver into a clean 500 ml volumetric flask. Rinse the pipet three times with

diluent solution, returning all rinses to the

flask. Dilute the flask to calibrated volume

with diluent solution and mix by inverting 30 times.

Rinse the pipet with water, condition with the

next unknown solution, and repeat steps 7 and 8 as many times as there are unknowns to be analyzed. (NOTE: If the rinse solution doesn't

28
completely wet the sides of the pipet, clean the

pipet by rinsing it several times in a solution

of aqua regia (HCl-HNO^ 3:1). Then, repeat

step 9 .

)

10, At the conclusion of the dilution procedures

there should be

:

a. One 500 ml volumetric flask containing a 1:50 dilution of the blank stock solution.

Label "B "

.

b. Three 500 ml volumetric flasks each containing 1:50 dilutions of the calcium stock

standard solutions. Label "4.00", "5.00",

and "6.00" (these are in meq Ca/1)

.

c. As many 500 ml volumetric flasks each con- taining a 1:50 dilution of each of the un- known solutions as there are to be analyzed.

Label appropriately. (NOTE: For the sake

of clarity in following steps it is assumed

there is one unknown labeled "x".)

29
Atomic Absorption Spectrometry (AAS) Measurement Procedure

It is assumed that the operator is fully familiar

with the instrument to be used. It is not possible in this

method to give detailed instructions necessary to assure instrument stability, linearity, flame conditions, etc.

In general, the accuracy of the method cannot be attained

unless the instrument is in optimum operating condition and

meets all the specifications set forth by the manufacturer.

Repeatability of readings of the same solution within ±0.5%

(maximum) is a necessary condition.

1. Instrument and Electrical Adjustment . Prepare the atomic

absorption spectrometer for operation according to instructions provided in the operator's manual. Place

the calcium hollow cathode lamp in the lamp housing

receptacle. Turn the power supply switch to "On". Select the optimum current for the lamp, and allow

ample "warm-up" time for the lamp to become stable.

Adjust the monochromator slit and set the wavelength

selector to the calcium resonance line at 422.7 nm

o or 4227 A . Adjust the photomultiplier dynode voltage

to give optimum current output with minimum dark current.

2. Flame Condition. Open the tank valves on the air and

acetylene supplies. Adjust the secondary regulators

as recommended by manufacturer. Check the burner

to make sure the premixing chamber and nebulizer are clean and free of any foreign obstructions. Insert

a three-slot (Boling) burner head on the burner. Light

the burner and adjust the air and acetylene flow rates

recommended for the instrument. To stabilize the

temperature of the burner head, aspirate water into the

flame for at least 10 minutes before proceeding to the

next step. (NOTE: A fuel-rich air-acetylene flame gives

optimum sensitivity for the measurement of calcium; however,

30
it may be difficult to obtain the precision specified

in this method with a fuel-rich flame. Therefore, it

is suggested that a stoichiometric or slightly fuel - rich flame be used to obtain the highest precision for

calcium in serum.)

Determination of Optimum Absorption . Determine the

stability and repeatability of the instrument as well

as the calibration curve as follows

:

a. Adjust the instrument to zero absorbance while

nebulizing water.

b. Nebulize the solution of 4.00 meq Ca/1 and. measure the absorbance.

c. If the absorbance is not 1.000 units, adjust the

scale expansion of the readout system until the

absorbance value is greater than 1.00 0. (NOTE:

A scale expansion of approximately 5 is required

for most instruments .

)

d. Readjust the instrument to zero absorbance with water.

e. Nebulize the reagent blank, 4.00, 5.00, and 6.00

meq Ca/1 and record their absorbances . Nebulize

water between each of the standard solutions and

check the zero value.

f . Repeat the sequence of blank and standards as outlined in 3e until a repeatability of readings

for the same solution is within ±0.5%

g. Subtract the absorbance value for the reagent

blank from the average value obtained for the

standard solution.

h. Plot on rectilinear graph paper the absorbance,

corrected for reagent blank, as ordinate, versus

the concentration of standard expressed inmeq/1.

A typical calibration curve is shown in Figure 1 of Ca as abscissa. (NOTE: If concentration

values are determined directly from the instrument
1.600 ~

1.400 -

< 1.200 -

SCALE EXPANDED - 5

/ / 1.000 -

I—^

0 4.00 4.50 5.00 5.50 6.00

Ca, meq/liter

Figure 1. Typical Calibration Curve for the

Atomic Absorption Spectrometry

Determination of Calcium

32
instead of the absorbance values , follow the same procedure.

)

i. If the calibration curve is not linear, prepare

calcium standards of 4.50 and 5.50 irteq/1 following

the procedure given previously.

4. Absorbance Measurements. Measure the absorbance of the

unknown solution as follows

:

a. Repeat the calibration curve as outlined in Step 3. b. Nebulize the unknown solution and then nebulize

the two standard solutions that are closest to the value of the unknown. Record these absorbance

values

.

c. Repeat this sequence of standards and a single un- known until 10 valid measurements have been obtained,

d. Repeat steps 4a-4c on additional unknowns and their

associated standard solutions

.

5. Valid Measurement . To obtain a valid measurement,

follow the sequence of standards and unknowns and

record the data as follows

:

4.00 4.50 5.00 5.50 6.00

Ca meq/1

No. Sample

1 Standards

2 Unknown ( 1

)

3 Standards

4 Unknown (2)

5 Standards

6 Unknown ( 3

)

1.084 1.222 1.355 1.486 1.608

1.477

1.354 1.480

1.475

1.351 1.488

1.420

1.320 1.475

Two valid measurements for the unknown in test 2 and 4 were obtained as the difference between consecutive standards is less than 1%. However, the value

obtained in test 6 is not valid because the difference

between 1.351 and 1.320 is greater than 1% E.g.

33
1.355 - 1.354 = .001 or <.1%

1.486 - 1.480 = .006 or .4% 1.354 - 1.351 = .003 or <.1%

1.488 - 1.480 = .008 or .5%

6. Calculations . Calculate the sample concentration

by an interpolation technique.

a. Mathematical Interpolation . Calculate the

concentration in meq Ca/1 for each valid

measurement using the following formula:

where C = sample concentration; meq Ca/1

s

l = concentration of lower standard, meq Ca/1

S

2 = concentration of upper standard, meq Ca/1

A

X

= absorbance of unknown

L S

l = absorbance of lower standard

S

2 = absorbance of upper standard

b. Graphic Interpolation . Plot the absorbance values

for the upper and lower standards versus concen- tration meq Ca/1 on rectilinear graph paper

for each set of data. Draw a straight line between data points and determine the concentration

of the unknown from the curve.

c. Computer Interpolation . By means of a least

square plot using the paper and lower standards,

compute the concentration of the unknown with a computer.

d. Compile the concentrations obtained by method

a, b, or c for the ten valid measurements and

determine the mean value. For the best precision

methods 6 a or 6c are recommended.
V. COMMENTS OF THE COOPERATING LABORATORIES

During the course of the five exercises and especially

at the meeting held at NBS in June 1971, the cooperating

laboratories made comments, suggestions, and criticisms.

It is not necessary to list each of these, but those which

had a bearing on the future exercises, or were adopted, or threw light on current steps in the procedures are given

below:

Lab A - Exercise 4 - Serum samples for this exercise

contained sufficient insoluble residue so that

reproducibility of aliquoting was seriously impaired.

Precision on separate aliquots taken 6 days apart

ranged from 0.3 to 1.7%;

Lab A - Exercise 5 - Only deviation required from the

recommended procedure was to rinse the pipet with

a solution of HCl-HNO^ (3:1) to assure proper drainage.

Lab B - Exercise 1 - Adequacy of rinsing the pipet

according to the procedure was questioned. The

greatest source of instrument error was thought to be in making the reading. Because the published Pybus

,

et al., method was designed to measure Ca in the presence of Na and K, these were added to the NBS unknown

samples. Auxiliary work showed a 1% lower result

when Na and K were absent. Suggested that in a future

exercise test samples be supplied with and without Na

and K to verify this finding.

Lab B - Exercise 2 - Mg(C

2 H30

2

) 2 added to standard solution, and internal reference to SRM used throughout.

Lab B - Exercise 4 - Two days work required to prepare

glassware and make up solutions.

.uab B - Exercise 5 (First set of data not used and rerun

made.) - Method run by senior technologist and not

professional chemists. Second run 1-2% higher than run 1. Significant unauthorized changes made in

35
procedure; e.g., three 5 ml pipets used instead of one 10 ml pipet; dilutions made were 5:250 instead

of 10:500; ambient temperature conditions not con- trolled. Furthermore, the technologist was not

enthusiastic about performing the work.

Work rerun by willing senior personnel who

followed protocol exactly.

Lab C - Exercise 1 - Protocol followed as faithfully as possible. Instrument problems experienced over past

3 weeks; occasionally high-noise level was observed.

A rather large variation between sets of samples in run 2 noted. It was difficult to adjust instrument

in mid-run and results were calculated relative to the immediately preceding 5 meq/1 standard.

Dilution technique was changed. A single class

A 50 ml volumetric pipet was used to deliver all the

diluent and a single class A 1 ml pipet used for

standards and samples. The 1:50 dilution does not

give satisfactorily high absorbance values to feel we are performing with the highest accuracy and precision of our instrumentation.

La

2

0 3 and Mg( c

2 H3°2^2 used were from different

suppliers than those cited in original candidate method.

Glassware was washed according to routine procedures prevailing in this lab: (1) soak for 24 h

in chromic acid, (2) rinse 4 times with deionized

H^O, C3) rinse 4 times with deionized distilled £^0,

and (4) air dry under cover. Lab C - Exercise 3 - Absorbance and concentration values were based on the average of 10 readings

.

Because of insufficient sample received, 3 samples were diluted 5:250 instead of 10:500.

The sera samples for this exercise with apparently

actively-growing cultures led to some difficulties

36
with pipetting. If large dilution volumes are to be

maintained, an anti-foaming agent should be used in

the future.

As a result of discussions at the meeting held

at NBS , June 19 71, there is some confusion concerning

the directions of the present protocol. Suggest it

be extensively modified and clarified to pin down

exact procedures to be used concerning "valid measurements" and rules for discarding data. Also the

order in which the sample, standard, blank, are to be run is not exactly clear in the present protocol.

Lab C - Exercise 4 - This lot of sera even worse than

those for exercise 3; sampling errors due to the

growth are possible.

In run 1 —2,0,0,1 sets of measurements rejected

as not meeting the valid measurement criteria. Then

5 sets rejected for the next sample, so the aspirator

was cleaned and a new sequence started with zero subsequent rejections. The rest of run 1 saw 1,2,2,1

and 2 sets rejected for the sample sequences remaining.

In run 2, for the five NBS samples run in duplicate, the following sequence of rejected readings was obtained: 1,3,1,7,3,1 entire set of 10 readings,

2,7,2, and 6 respectively, for the corresponding

samples sequences.

Lab D - Exercise 1 - Dilution procedure used 1 ml pipet

to 50 ml volumetric flasks. Calibration curves are not exactly linear over the range 4-6 meq/1. Results

are calculated from a calibration curve constructed

for each batch. Curve shows small variations from

batch to batch. Standards for calibration are run

in duplicate followed by quality control specimens

.

Patient specimens are run in groups of five followed

by 2 standards used as drift markers. Changes in

these outside + 1% is the criterion for recalibration

.

37
No Sr internal standard used. No blank solutions

used since measurements made a relative basis, i.e.,

sample absorbance is made relative to absorbance of

calibrating standards.

Lab D - Exercise 2 - Protocol calls for a double rinsing

of pipet and 10 ml sample size. With 2 runs to be

made there is insufficient sample. Suggest a partial

filling of pipet (for rinses) and then inversion.

Plard to follow protocol, especially as to deadlines (schedules) because the work must be accommo- dated to an extremely busy analytical system.

Samples received for exercise 3 not considered

fit for analysis; had sediment and floating particles.

Ten replicate readings not provided as normal

procedure is to run at steady state, which is 2-3

minutes. The steady state achieved (shown on recorder

chart attached) indicates the variation that would be

expected if interrupted replicate determinations had

been made.

Lab D - Exercise 4 - Due to routine work load, repeated

sets as described in the protocol were not able to be run. Duplicate readings provided from chart re- corder readout system.

In both rounds 1 and 2 , two sets of data were

rejected.

Lab D - Exercise 5 - New burner assembly unit installed.

Calibration curve is now linear.

Lab E t Exercise 1 - Reagents required by the protocal

not arrived. Ran exercise using our present method

based on the Trudeau and Freier method [6]

.

Lab E - Exercise 4 - (Exercise aborted) -Quantitative

transfer of turbid and contaminated solution was most difficult due to clogging of the pipet and the

flocculent debris that adhered to the inner surfaces.
Visible particles observed in the final diluted solutions. Protocol followed exactly but instrumental

precision required by the protocol could not be

obtained. Decided to abort remainder of exercise.

Physical and instrumental problems should be

alleviated when we move to new quarters.

Lab F - Exercise 1 - Instrument location is not ideal;

flame is subject to drafts. Attainment of stable

meter readings has always been something of a problem. For each run, standards from lowest to highest

concentration were run, followed by the unknowns

in assigned order. Baseline was set with diluent con- taining La and Sr and checked and reset, if necessary,

after at least two sample readings.

Readout indicator was calibrated in percent absorption and read to nearest hundredth although move- ment of the dial several units in either direction often had no noticeable effect on the meter needle.

Graphical plot of points on calibration curve

was not precisely linear, and best fit standard

curve was computed. Values of unknowns for three

ranges of standards were computed directly, and were compared with values taken directly from the calibration curve. Differences of 1-3% were noted. This

raises the question of what is the proper procedure

for calculating the unknowns

.

Unknowns and standards were prepared the day

before the run was made. A 1 ml pipet was used and

rinsed with deionized H^O rather than saline. Wo particular preparation of the instrument made, e.g.,

burner head not cleaned. A different batch of diluent

prepared for run 2. Lab F - Exercise 2 - Protocol for standard values bracketing the unknown to be within 1% of each other. The

3y
maximum difference between standard readings over a series of 10 determinations (in a set on one unknown) was 3.1%.

Each pair of standard readings used to bracket

the unknown was averaged and the average of the 10 readings used to compute the reported value.

Lab F - Exercise 3 - In order to verify matrix effects,

both Mg(C

2 H3 02

) 2 and SrCl

2 were omitted from diluent

and stock standard solutions. Slope of the standard

curve fell from ^45° to one-half that.

Deviation from the protocol is noted. It was difficult to maintain the % absorption values for

the standard over the 5-hour period of the run. With

care this could have been done, but would have doubled

the time devoted to the exercise. Nevertheless, the two standard values used to bracket the unknowns were

always reproduced to approximately ±1% of each other.

The method of calculation was thus modified from that

of picking unknown values from a standard curve to that of ratioing the unknown reading to that of the

average reading of the bracketing standards and multiplying this factor by the known standard value.

Are other labs having difficulty in maintaining

a stable standard curve throughout the run?

Lab F - Exercise 4 Our instrument has no better than a 30% chance of any pair of readings reproducing within

±0.5% (revised protocol requirements). We will pass

for this exercise.

Lab H - Exercise 5 - The need to rerun Exercise 5 has led

us to investigate our glassware washing procedures

.

We found some white residues on several volumetric

flasks. Tap water may still be in some flasks before

drying, since deionized water rinsing is a hand operation. For the rerun we pre-rinsed flasks with deionized water and diluent before use.

40
Within run precision is better than run-to-run

agreement.

Lab K - Exercise 3 - Followed by Pybus , et al., method,

except no integration used; internal standard used.

Instrument extremely stable; readings obtained directly.

Unable to complete every aspect of this exercise (due to time limitations) and values reported are not based

on 10 independent readings

.

Lab K - Exercise 4 - Instrument overhauled after completion

of Exercise 4. Found some dirt-film on internal optical surfaces. After cleaning sensitivity increased

and noise level decreased.

Sample #564 had fibrin clots.

41
VI . RESULTS

(John MANDEL

)

A . Introduction

The data in this report cover five separate "exercises",

each of which is an interlaboratory study conducted at 3 to 5 levels of concentration. In the course of running these five

exercises, a number of modifications were introduced, both in

the basic protocol and by the individual laboratories in their

equipment, personnel and procedure. Furthermore, not all laboratories participated in all five exercises. These circumstances make it undesirable to run a single overall analysis

covering all five exercises. Instead, individual analyses were

run on the data for each exercise, and subsequently, the data

for each laboratory were studied separately over the period

covering all five exercises. The objectives of the statistical

analysis were as follows

:

1. Find measures for within and between laboratory

variability for each exercise.

2. Determine deviations from the "target values" (in this case, the values given by the isotope dilution

.method) for all measurements by all laboratories,

Study the pattern of these deviations, both between

laboratories within each exercise, and between

exercises, for each laboratory.

The first objective covers precision ; the second, essen- tially accuracy

B. Summary of Precision and Accuracy Data

Table 5 is a summary showing, for each solution in each

exercise, the variability between duplicates (same laboratory,

same run) , between runs (same laboratory) , and between laboratories. The table also shows the bias, i.e., the difference

between the average value for all laboratories and the isotope

dilution value, for each solution, expressed as percent of the

42
isotope dilution value.

The precision data are to be interpreted as follows: For

"replicates", the statistical population considered is a hypothetically infinite set of replicate values obtained by

any given laboratory in a single run. For "runs", the

population is a hypothetically infinite set of values made

up of single determinations, all from the same laboratory,

but each from a different run. For w labs ! ', the population

is a hypothetically infinite set of values made up of

single determinations, each from a different laboratory.

In each case, the measure (%CV) is the estimated standard

deviation for the population considered, expressed as percent of the isotope dilution value.

A more detailed presentation of the data is afforded

by the graphs shown in Figures 2 to 16. These will be discussed further in this report, following the discussion of

the Summary Table.

43
C . Discussion of Summary Table

From the last column of Table 5 it is apparent that on

the whole, the atomic absorption method, as carried out by

the laboratories participating in this study, gave slightly

lower values than the isotope dilution method. Only two of

the nineteen bias values are positive, and both of these are

small. It is also apparent that with the exception of Exercises 3 and 4, both of which used questionable serum solutions,

the bias is generally small compared with the variability

between laboratories. Thus, while a small negative bias appears

to be present in the atomic absorption method, this bias is

essentially negligible compared with the larger random errors between laboratories

.

The summary also shows a noticeable effect of runs, es- pecially for the serum solutions (Exercises 3, 4, and 5).

Such a situation is common in analytical chemistry: measurements made at several days ' interval generally show less

agreement than measurements made in replicate at essentially

the same time

.

Finally, laboratories show measurable systematic differences from each other, as evidenced by the fact that the co- efficient of variation between laboratories (Table 5) is

practically always larger than that between runs. Again this

effect is slight for aqueous solutions (Exercises 1 and 2)

,

but more noticeable for serum solutions (Exercises 3, 4, and

5) . In evaluating the summary data, it must be kept in mind

that the coefficient of variation is related to a one-standarddeviation effect. One can expect a range of ±1.96 standard

deviations to cover approximately 95% of all measurements.

Thus, to cover 95% of the population of measurements in

each category, the coefficients of variation in Table 5 should be multiplied by 1.9 6 and allowance should be made

for that variation in both directions.

44
Elsewhere in this report, the vicissitudes of the participating laboratories in the course of this study are reported

in detail. From these it follows that reasonably satisfactory

and stable conditions, both in the sample preparation (of

serum) and in the laboratories 1 techniques , were not achieved

until Exercise 5. The data in the summary table show that

even then, as much as 2 to 4% overall range of variation over

| the various laboratories is likely to occur.

! D. Graphical Report of Results

Figures 2 to 6 show the individual results for all participating laboratories for each of the five exercises . The

plotted values are the deviations of individual measurements*

from the corresponding isotope dilution values. For each

laboratory, ten values for run 1 are plotted first for all

I solutions, followed by the values for run 2. Figures 7 to 14 contain the same data, but each figure

now represents a single laboratory and the values are plotted

in order of successive exercises.

For purposes of comparison, the results of the isotope

dilution method are presented in similar graphical form in

Figures 15 and 16 in which each point represents a single

determination. For Figure 15, dealing with the aqueous

solutions, the deviations are calculated with respect to the

value provided by the known weights of calcium, salt and water.

In Figure 16, dealing with serum solutions, the deviations

are calculated with respect to the average value of the isotope

dilution determinations. Thus, Figure 16 provides information

solely on the precision, whereas Figure 15 provides information

also on the accuracy of the isotope dilution method.

Figures 7 to 14 essentially confirm the conclusions stated

*except for Exercise 1, in which averages of several measurements are plotted. The design for Exercise 1 was slightly

different

.

45
above. In addition, they allow us to follow the events as they occurred in the various laboratories in the course of

this study. If we exclude Exercises 3 and 4, because of

the unsatisfactory condition of the serum, we find that only

4 laboratories (A, B, C, and E) stayed consistently within

a band of ±2% deviation from the isotope dilution value.

With great care, both in the preparation of the sample, and

in the laboratory technique, it appears that a goal of ±2%

overall error could be achieved. The data also show that

any significant departure from these conditions of constant

concern and control will result in much larger errors

.

46
TABLE 5. SUMMARY OF PRECISION AND ACCURACY RESULTS

Isotope Exercise Solution

No

.

Di lut ion Value Replic

.

%C . V

.

Rmq (b) Runs Taho (c) Labs Bias

1 1 4.0783 1.1 1.1 1.1 - .02

2 5.1816 .7 .7 1.0 - .30

3 5. 8673 .9 1.0 1.2 - .76

2 1 3. 5928 1.3 1.4 1.7 .09

2 4. 8922 .8 1.3 1.7 - .10

3 6.3767 .9 1.4 1.5 - .63

3 2 4.5709 .4 .8 2.7 - .79

4 4.8303 .8 1.2 4.0 -1.62

3 5.0249 .4 .6 3.9 -1.75

1 6.1526 . 3 1.0 2.2 -2.28

4 3 3.6228 .6 1.4 1.6 - .15

1 4.1118 .2 .6 1.1 - .17

2 5.0598 .2 .4 1.0 - .93

5 5.5650 .2 . 4 .9 -1.20

4 5.7934 .3 .5 1.6 - .93

5 1 3.5704 .3 1.3 2.2 .24

2 4.2939 .5 1.0 1.4 - .48

3 5.0234 .2 .5 .7 - .13

4 5.7325 .2 .4 .8 - .18

(a) Percent coefficient of variation for replicates within runs.

(b) Percent coefficient of variation for determinations from different runs.

(c) Percent coefficient of variation for determinations from different laboratories.

(d) Difference between average of all labs and ID-MS value, as % of IDWyiS value.

47
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53
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54
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55
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56
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57
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- 1 1 - S 0

-1

l! I, tt

1 n i

1

_ oL -

2 -3

-4 1

1

in ii i i Mil' | | Mill Mill MM MM

ROUND 1 2 1 2 i 2 12 12

EXERCISE 1 2 3 4 5

Figure 12.

58
4h

3

21-

LABORATORY H

PERCENT DEVIATION FROM ID-MS TARGET VALUES

TT

III III III III I I I I llll I II I I I I I I I 12 12 12

EXERCISE i 2 3

I 2

ii ii 1

1

I 2

5

Figure 13.

59
LABORATORY K PERCENT DEVIATION FROM ID- MSTAIWET VALUES

-

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 n in

1

"i in ii i

1

in i

1

ROUND 12 12 12 12 12

EXERCISE 1 2 3 4 5

Figure 14.

60
61
cz> CVJ U_l

I I N0I1VIA3Q %

62
VII. THE ISOTOPE-DILUTION MASS SPECTROMETRY WORK

(Larry J. MOORE)

A. Theory of Stable Isotope Dilution Analysis

The analysis of trace level components by stable isotope dilution mass spectrometry is a well-established technique and has been successfully applied to a wide variety

of materials over the last three decades. Any analytical

method capable of measuring the relative quantity of stable

isotopes in an element is a potential candidate for isotope

dilution analyses; however, mass spectrometry has been used

almost exclusively due to its sensitivity and accuracy. More

specifically the thermal ionization technique has been the

predominant mass spectrometric method used because of its inherent accuracy, sensitivity and versatility for the measure- ment of inorganic elements.

Isotope dilution analyses are performed by measuring

the change in the relative magnitude of two analyte isotopes

when a measured amount of a material enriched in one isotope

is added to the sample. The method of calculating the concen- tration of the analyte from an isotope pair ratio measurement

is shown in equation 1

.

Concentration, ppm (wt) = Wsp

C ^ A

sp R B

sp^ M_ (1) BR - A Ws Weight of spike solution, grams

Concentration of spike, ymoles/gram of solution

Atomic fraction of isotope A in spike

Atomic fraction of isotope B in spike

Atomic fraction of isotope A in sample

Atomic fraction of isotope B in sample

Experimentally measured ratio

Atomic weight of analyte

Weight of sample, grams

sp

sp

J sp

63
Upon dissection of this equation it is observed that a successful and accurate isotope dilution concentration meas- urement must include the following steps:

(1) Addition of the spike to the analyte, chemical

dissolution and (usually) treatment of the sample

to eliminate potential mass spectrometric interferences . (2) Accurate measurement by mass spectrometry of the

isotopic composition of both the analyte as it

exists naturally in the sample and the enriched

isotope mixture used for altering the natural

isotope ratio.

(3) Determination of the concentration of the spiking

isotope solution by calibrating it versus accu- rately known amounts of a solution of the "natural1 analyte

.

(4) Determination of the amount of natural analyte

contamination (blank) incurred during the chemical

processing

.

(5) Measurement of the altered isotope ratio and

calculation of the concentration.

B. Mass Spectrometric Isotope Dilution as an Absolute

Analytical Technique

1. Systematic Errors Possible in Isotope Dilution Measurements

a. Chemical contributions

A critical evaulation of the errors possible in

the various chemical treatment steps results in several

contingencies. The two most frequent forms of systematic

error in chemical processing are (1) incomplete dissolution

of sample and (2) the chemical blank or contamination incurred during the processing.

For concentrations where the chemical blank represents

a significant fraction of the total analyte, the blank may

64
become a contributing error. In fact, when all the other

contributing systematic and random errors of a total isotope

dilution method are under control the blank may become the

limiting error.

The blank can be dissected as consisting of contributions from reagents and particulate fallout . To reduce

contributions from the first source, ultra-pure acids (Ca

present in HC10

4 , HN0

3 and HC1 at 0.2 ppb-ng/g-0.2 ppb and

0.06 ppb, respectively) are prepared and used at NBS . To reduce particulate contributions all the chemical preparations

are carried out in a Class 100 clean air hood inside a vertical

flow clean room [7] . If repetitive blank determinations for a method are un- certain to <^50% the blank can be treated as a systematic error whose uncertainty as a correction is equal to the randomness

of its measurement. Or, if the blank is uncertain to >^100%

then the total blank may become the limit of error for trace

concentrations and in that case must be applied to the concentration result as a plus or minus limit of error. Of course,

the error from a given blank level becomes larger as the analyte

concentration becomes lower. The contribution of the blank

correction to the total error therefore depends on (1) the con- centration of the analyte, (2) the reproducibility of the

blank, and (3) the magnitude of competing error contributions

from mass spectrometric and other chemical sources , as described in this section. The importance of recognizing the

random blank as a basic limit of error in many trace element

analyses, regardless of the analytical technique, cannot be

overemphasized. Its identification, control and correction

should be considered an integral part of the analysis.

In addition to incomplete dissolution of the sample,

there are other sample-oriented errors that can also occur

if reasonable care is not taken by the chemist in sample

preparation

:

65
(1) isotope fractionation occurring during non- quantitative sample recovery, (2) incomplete mixing

of the sample with the separated isotope, and (3) partial loss of the analyte upon sample dissolution.

As seen in equation 1, the accuracy of the determination

is directly dependent on the accuracy of the spike calibration. As the calibration uses a known "sample"' most of the

sample-oriented errors apply here as well.

If the possible errors outlined above are considered in

the careful design and application of an experiment, they can

be eliminated as sources of systematic bias.

b. Mass spectrometric contributions

The formation of ions in a triple filament thermal

ionization source is accomplished by heating the sample on each of two 1x30 mil rhenium ribbons to volatilize the

sample to the relatively hot surface of a third rhenium

ribbon where a portion of the atoms leave the surface as ions. The ionization process is a very gentle one, resulting

in a distribution of excess ion energies of only a fraction

of an electron volt, which is an order of magnitude less

than its closest competitor, the electron impact source. The

low energy spread of the ions and the stability of the ion

beam are the bases for the inherent capability of the thermal

ionization technique to make accurate isotope measurements.

Errors in the measurement of isotope ratios can occur

from several sources. The ability of the operator to con- trol fractionation of the analyte isotopes in the ion source reproducibly from one sample to the next is usually the

limiting error in isotope dilution analyses. In the case of

40 4 4 Ca/ Ca measurements, the difficulty in controlling isotope

fractionation is compounded by a basic limitation in the

measurement of isotopes involving large mass differences

(difference of 10% for 40 Ca and 44 Ca)

.

66
Instrumental errors can occur in the non- linear measurement of varying isotope ratios. However, the instruments

used in this work have been repeatedly shown to be biasfree in the measurement of ratios varying from 1/20 to 20/1

at a 95% limit of error (calculated as to) of ±0.05% [8].

Thus any contribution from this source is negligible since

40 44 the Ca/ Ca reproduces only to ^±0.2% (95% L.E.), which is

a factor of four higher than ±0.05%.

Background contributions from the filament material at the mass positions of interest (in the present case, 40 and

44) can be a source of error and must be investigated.

Interferences from isobars at the mass positions of

40 40 interest can be a problem (e.g. K is isobaric with Ca)

and must be controlled. Usually the sample is processed

chemically to rid it of isobaric interferences, although

occasionally the interference can be eliminated during mass spectrometric analysis.

Particulate contamination can occur during loading of

the sample prior to mass spectrometric analysis. Sample

loading is therefore carried out insofar as possible inside

a Class 100 clean air hood and care is taken to avoid excess handling of sample-holding equipment.

c. General considerations

When the potential sources of systematic bias

are eliminated, the residual error is composed of the random

errors associated with the measurement of the isotope ratios

and with the chemical preparation steps. If the blank

correction is sufficiently large and random, then it must

compete with the total random error derived from the other

sources to become the limiting error for the analysis. With

these qualifications, mass spectrometric isotope dilution

analysis then becomes a method for the absolute analysis of

trace elements in a variety of matrices.

2. Historal Performance of Isotope Measurements
Because of its inherent accuracy, thermal ionization

mass spectrometry has found extensive application at NBS

for the measurement of isotope ratios. In the process of

making these measurements the Analytical Mass Spectrometry

Section has invested, conservatively, 20-30 man-years in the

elucidation of the various error components. The end result

of these efforts has been the absolute determination of the

atomic weights of Mg, Pb, Cr, CI, Ag, Rb, B, Cu and Br [9-17]

In addition, K, Ni , Sr, Re and Si are currently under develop

ment for atomic weight determinations.

Over the last few years the knowledge acquired during

these isotope ratio measurements has been applied widely to isotope dilution analyses. As an example, the certification

of the Trace Elements in Glasses standards has required the

development of isotope dilution analysis methods for U, Th,

Fb, Tl, Rb and Sr among others at concentration levels of

500-0.02 ppm. Accuracies for some elements in these standards have been maintained at <^0.25% (95% L.E.), even at concentrations less than 1 ppm (publications in preparation)

.

Applications of isotope dilution have also been made to a wide variety of other matrices, from beef liver and orchard

leaves to nuclear fuel rods and reference steels. In all

these applications the rules by which isotope measurements

are made have not been found to change, and are directly

applicable to the determination of calcium in serum.

C . Analytical Procedure

1. Chemistry

The individual samples for analysis by atomic absorption in Exercise 1 were taken from stock solutions of SRM

915 CaCO^ which were prepared at concentrations of approximately 80, 100 and 120 ppm calcium. Aliquoting of the

samples was accomplished by transferring 50 ml portions of

the stock solution to 100 ml borosilicate glass ampoules

followed by sealing of the ampoules . No error from weight

68
loss was observed due to evaporation of the solution during

the sealing process

,

Ampoules from the beginning, middle and end of the

aliquoting procedure were taken for isotope dilution analysis

and their pH and density were measured. Samples were obtained from the ampoules by multiply extracting 5 gram

quantities with a syringe, which were then accurately weighed.

44 These samples were spiked with a known amount of the Ca

separated isotope and converted to the nitrate form for

isotopic analysis.

Solutions for Exercise 2 were prepared and ampouled as in the first exercise, except that sodium and potassium were

added prior to ampouling at 140 mg/1 and 5 mg/1, res- pectively, to simulate the ionic strength of serum The

addition of the alkali elements required that the samples

taken for isotope dilution be processed by ion exchange to

40 40 remove the potassium, since K is isobaric with Ca. Otherwise the isotope dilution samples were processed as in

Exercise 1. To obtain atomic absorption samples for Exercise 3, 50 ml aliquots were transferred to 100 ml ampoules from four 1- liter lots of pooled blood serum supplied by Hartford Hospital.

Multiple samples for isotope dilution were withdrawn by sy- ringe from selected ampoules and HCIO^ was added to destroy

the organic matter present in the serum. The calcium was purified by ion exchange and the isotope ratios determined

by mass spectrometry.

Exercise 4 was carried out as in the third exercise,

again with pooled serum samples supplied by Hartford Hospital.

iio significant changes in the chemical procedure were re- quired, except that a larger bore syringe was used to avoid

sampling problems described later.

The aliquoting, sampling and analysis in Exercise 5 were performed as before, but with samples supplied by the Center

69
for Disease Control.

Insofar as possible, all of the above chemical operations U were carried out in a Class 100 clean air hood. Following

the construction of a vertical flow clean room midway through

the series of analyses all chemical operations were transferred to this facility.

2. Mass Spectrometry

All calcium isotope ratio measurements were made using

three magnetic sector, single focussing thermal ionization mass spectrometers: one was a 12" radius of curvature, 68° sector instrument, and the remaining two were identical 6" radius of curvature, 68° sector instruments. All the instruments use identical ion sources and deep faraday cup

collectors 118-19]

.

Care was taken to maintain an identical heating and

fractionating pattern for the analysis of both standards and

samples to ensure no systematic bias from this source.

40 Filament material background contributions from Ca and

40 interferences from K were carefully investigated and removed.

Calcium samples and standards (SRM 915) were loaded as calcium nitrate onto each of two rhenium sample filaments.

Using a triple filament rhenium technique (the details of the

chemical and mass spectrometric techniques used in the analysis

of Ca by isotope dilution are being published separately) , the

40 44 samples were analyzed for their Ca/ Ca ratios, and the

concentrations calculated according to Equation 1. D . Analysis Results

The following compendium of data is composed of the

isotope dilution concentrations for Exercises 1-5, expressed

as milliequivalents of Ca per liter. As all of the isotope

dilution calculations in the Analytical Mass Spectrometry

Section are performed on a weight basis; i.e., ppm (wt) , the

density was carefully measured for each concentration level

70
in each exercise to permit a conversion of the ppm concentrations to milliequivalents/liter:

Concentration, milliequivalents/liter = - PP1"),,, ^

ppm = Calculated concentration from Equation 1, yg/g

D = Measured density, g/ml

EW = Equivalent weight of calcium (20.04).

Exercise 1 (Table 6) Exercise 1 was the first attempt of the Analytical Mass

Spectrometry Section at the isotope dilution analysis of

calcium. Two mass spectrometers were used, each operated by

different analysts using the same mass spectrometric technique.

40 44 Ca/ Ca isotope ratio representing the average of 10 ratio

measurements was determined for each sample, and the average

ratios that produced outlying concentration values were

repeated. Because these solutions were prepared using primary

chemical methods, the agreement among the aliquots for a given

concentration level represents a direct measure of the reproducibility of the isotope dilution technique.

Using 16 degrees of freedom, a 95% L.E. was calculated

for each operator's concentration values*. The magnitude and

variation of the operators' measurement uncertainties (0.35%

and 0.22%) are due to the fact that the analysis and the

development of an analytical technique for calcium were

carried out concurrently. formally a technique is investigated

and developed over a period of several months , and only then

is the perfected technique applied to accurate isotope measurements .

*The ratio of individual concentration-to-average con- centration was computed for each sample for each concentration

level and the variations from 1.0000 were used for calculating

the 95% L.E. for a single analysis.

71
From previous experience, it has been found that two

operators are often useful in establishing the absence of

any systematic bias introduced by the operator. Although

there was no detectable operator bias indicated in the

measurements, the average concentration values of each operator were averaged to obtain the final reported values . The agreement between the experimental and calculated values may be

taken as an experimental verification that the possible sy- stematic errors have been eliminated and that the calculated

confidence limits are a reflection of the random errors as- sociated with the isotope ratio measurements.

The remaining possible systematic error is that of the

blank, which was found to be reproducible at <^0.1% of the

total amount of calcium per sample. Again, the excellent

agreement between calculated and experimental values indicates that the blank is insignificant as a source of

systematic bias.

Each sample number in Exercise 1 (1, 1A, etc.) re- presents a separate chemical preparation. In this exercise

three ampoules from each concentration level (e.g. vial numbers

1, 20, 120) were sampled in duplicate (1, 1A, 20, 20A, etc.).

Although not all the prepared samples in later exercises were analyzed for isotope ratios , the same sampling no- menclature is followed throughout Exercises 1-5.

72
Table 6. Concentration of Calcium in Synthetic

CaCO^ Solutions, meq/liter.

Exercise 1. sample Operator 1 Operator 2 Average Calculated Relative

11A

20

20A

120

120A

Average

4.093

4.072

4.071

4.066

4.071

4.075

4.0 75

4.0 83 4.089*

4.0 83 4.081

4.075

4.073

4.081

4.088

4.081

4.077

4.074

4.073

4.074

4.078 4.083 0.12'

21 5. 173

21A 5. 174

61 5. 180

61A 5. 176

80 5. 179

8 OA 5. 192

Average 5. 179

41 5. 867

60 5. 859 60A 5. 867

99 5. 875

99A 5. 852

Average 5. 864

95% L.E. ±0.35% C

5.180 5.177

5.183 5.178

5.187 5.183

5.180 5.178

5.183 5.181

5. 192 5. 192

5.184 5.182 5.186 0.08

5.874 5.871

5.876 5.868

5.870 5.869

5.865 5.870

5. 867 5. 859

5.870 5.867 5.867 0.00

±0.22% C

a Isotope ratios determined in duplicate.

k Relative error is the difference between experimental and

calculated value expressed as a percentage of the

calculated value.

Q Error expressed as 95% L.E. for a single analysis.

73
Exercise 2 (Table 7) These results were obtained using a single mass spectrometer, and involved the measurement of a smaller number

of aliquots for each concentration level. A 95% L.E. was calculated for the results in Exercise 2 in a manner

analogous to that of Exercise 1. Even though fewer degrees

of freedom were used the uncertainty for the measured

concentrations was less than for Exercise 1, as a direct

result of increased experience in the isotope measurements.

Also of significance in this exercise, the extra

chemical steps involved in the separation of calcium from

sodium and potassium resulted in no measurable introduction

of a systematic bias. A proof of the absence of any new systematic bias is reflected in the agreement (<^0.1%) between calculated and experimental concentrations. The blank

correction for this series was <^0.1% of the total amount of

calcium per sample.

In summary, Exercises 1 and 2 provided a testing ground

for experimentally demonstrating the validity of isotope dilution as a technique for the absolute analysis of calcium at approximate serum concentrations. By carefully controlling

and eliminating the well-known bias factors, the concentration

of calcium was determined with a consistent accuracy of

<^0. 1%. Exercise 3 (Table 8) A direct and successful extrapolation of the isotope

dilution technique to serum samples at the desired accuracy

level of <0.5% was precluded in this exercise by poor samples.

"Upon thawing, the several lots of serum succumbed to bacteriological action that resulted in turbid serum samples with

differing amounts of suspended particulate matter of varying

size. As a consequence, sampling by weight with a relatively narrow bore syringe was an apparent source of sampling error.

The syringe had the effect of filtering out all particles too large to be admitted into the syringe needle.

74
Table 7. Concentration of Calcium in Synthetic

CaCO^ Solutions (Na and K added) , meq/liter

Exercise 2.

Sample Isotope dilution

231 3.593

235 3.591

276 3.594

Average 3.59 3

Calculated

3.590

Relative error

0.0 8

C

215

215A

242

242A

Average

4. 895

4. 896

4. 891

4. 887

4.892 4. 887 0 .10 203A

263

266

Average

6.374

6.369

6.388

6.377 6.375 0.03

95% L.E :0.20%"

Relative error is the difference between experimental and

calculated value expressed as a percentage of the

calculated value.

Error expressed as 95% L.E. for a single analysis.

!

75
The ability to measure calcium concentrations by isotope

dilution with a 95% L.E. per analysis of ^0.2% can be inferred from Exercise 2. In addition, the uncertainty of the

concentration values of this exercise was diminished by making

a minimum of duplicate isotope measurements per sample.

Therefore, any significant excursions from the 0.2% accuracy

level were largely attributable to sampling. Although not

all of the data obtained in Exercise 3 are shown here, it was readily apparent that serious aliquoting errors occurred at the 1-3% level. The largest error of ^3% was in the 30 8-316

samples, while the remaining levels exhibited ranges <^1%.

Since the dominant error on this exercise was due to poor sampling, which in turn varied among the lots of serum, the error can only be estimated as plus or minus the range

of aliquots for each concentration level.

Exercise 4 (Table 9) To circumvent the sampling problems encountered in the

previous exercise a large bore syringe was used for sampling

and the serum was kept frozen until just prior to use. In general, the results were much more encouraging. Although

apparent sampling problems still existed in aliquots 561-576

and 501-516, the remaining aliquot groups (521-535 and 541-

554) ranged <^0.3%. An aliquoting error is easily seen in

516C and 516D, which are duplicate subsamples of one aliquot for the 501-516 group. Aliquots 501 (C and D) and 50 8 tC and D) agree internally and with each other to <^0.25%,

but differ collectively from 516 (C and D) by ^1%. The

blank correction was <0.1% for all samples.

Again, due to poor sampling, the error could only be

estimated as plus or minus the range of aliquot values per

concentration level.

76
Table 8 Concentration of Calcium in Serum,

meq/liter

(Exercise 3.

)

Sample Isotope dilution

301 6.094 a 308 6.079

316 6.288b

Average 6.154 ±3.40% c ' d

321 4.560

328 4.570

333 4.575

Average 4.568 ±0.33%°

341 4.999

348 5.020

356 5.056

Average

361 4.818

369 4.808

377 4. 859

Average

All isotope ratios determined in duplicate

(except 316)

.

Isotope ratio determined in quadruplicate.

Errors estimated as plus or minus the range of

individual aliquots for each concentration

level

.

Sampling is the dominant error for the results

in Exercise 3. The 95% L.E. for the isotope

dilution analysis of calcium is ±0.20%, as shown in Exercise 2.

77
Table 9. Concentration of Calcium in Serum meq/liter.

CExercise 4) Sample Isotope dilution Sample Isotope dilution

521-C 5.065 541-C 3.616

521-D 5.050 a 541-D 3.626

528-C 5.055 548-C 3.621

528-D 5.065 548-D 3.621

535-C 5.065 554-C 3.616

535-D 5.065 554-D 3.626

Average 5.061 ±0.30%b/C 3.621 ±0.28%b

561-C 5.801 501-C 4.106

561-D 5.785 501-D 4.096

568-C 5.775 508-C 4.096

568-D 5.765 a 508-D 4.096 a 576-C 5.806 516-C 4.136

576-D 5. 821 516-D 4. 142 a Average 5.792 ±0.97%b ' c 4.112 il.12%1

a Isotope ratios determined in duplicate.

b Errors estimated as plus or minus the range of individual

aliquots for each concentration level.

Sampling is the dominant error for the results in Exercise

4. The 95% L.E. for the isotope dilution analysis of

calcium is ±0.20%, as shown in Exercise 2.

78
Exercise 5 (Table 10)

The results for this exercise represent the quintessence

of our efforts toward an absolute serum analysis, for several

reasons

.

The Center for Disease Control supplied bovine serum

samples that were processed for homogeneity and protected

against bacteriological action by the addition of inhibiting

agents. Sampling error therefore was not a significant

problem in this exercise. An indication of the homogeneity

of the samples was observed in the density determinations,

4 which varied only one part in 10 over all four lots of serum.

I In Exercise 3 independent density measurements fluctuated by

as much as 0.1% for the individual lots of serum, and by

^0.5% between lots.

In any development work the opportunity to refine various aspects of an analytical technique always results in an

I eventual improvement in the resultant data. Through small

refinements in the mass spectrometric technique such as sample

loading and more stringent control of isotopic fractionation,

and in the chemical techniques such as better blank control

I and sampling procedures, the total technique was honed to peak performance. These refinements are reflected in the

data for this exercise.

Sample groups 621-625 and 605-609 probably exhibit the

ultimate accuracy attainable by this technique. In fact, the

agreement is actually better than the present overall ability

to consistently reproduce the calcium isotope ratios.

Duplicate isotope ratios on outlying values in groups

660-669 and 641-645 reproduced at <^0.25%, which approaches

the value for the random isotope ratio measurement error as calculated in Exercise 2. In fact, the 95% L.E. for the data

; of this exercise was calculated as 0.21%, which is in excellent agreement with the 95% L.E. of 0.20% for Exercise 2. However, the average of the individual aliquots should be

closer to the "true" or absolute value, as was the case in
Exercises 1 and 2 where the "true" value was known.

In addition to the above examples of a refined tech44 nique, the three Ca spike calibrations agreed to <0.0 8% and the blank correction was <0.03% for all the samples

analyzed.

80
Table 10 . Concentration of Calcium in

Serum, meq/liter.

CExercise 5 . ) Sample Isotope Dilution

621 3.569

624 3.573

625 3. 569 Average 3.570

605 4.294

608 4.293

609 4.294

Average 4.29 4

660 5.015

662 5.023

66 9 5.032 a Average 5.023

641 5.728

643 5.742 a 645 5. 729

Average 5 . 733

95% L.E. ±0.21%b

Isotope ratios determined in duplicate.

Error expressed as 95% L.E. for a single analysis.

81
VIII. DISCUSSION

Because the candidate method [4] had been so carefully

investigated, it was not thought necessary by the Experts

Committee to reinvestigate the factors that formed the basis

of that work. Therefore, there was no attempt made to re- study such factors as: interferences, reagent purity,

recovery studies, comparison with other methods, effect of

acids, etc., all of which were adequately covered by Pybus

,

et al. , in their work. Indeed, the conclusion by those authors

was that accuracy was limited solely by the achieveable precision of the method. However, during the course of that

work it became apparent that many parameters had to be closely

controlled and special attention was paid to these during the course of these studies. Perhaps the greatest unknown factor

was what contribution introduction of spectrometers (other

companies, other models) other than the type and model used

by Pybus, et al., would make to the inaccuracy of the system.

The other factors are: analytical chemical techniques

Caliquoting, pipetting, weighing, etc.;, sample condition,

quality and preparation of glassware, reagent quality (including water), significant deviations from the protocol,

and recording and reporting of data. All these will be

discussed in turn. A . Instruments

1. Balances

Although no data was collected on the actual

instruments used, the question was raised during

the June 19 71 meeting whether all laboratories

could weigh accurately to 0.1 mg. No exceptions were noted. With modern balances now readily

available to weigh to this degree of accuracy, no further discussion was felt necessary, and it was assumed that all weighings would in fact be so made. Furthermore, the directions for drying the

82
CaCO^ SRM are given on the Certificate of Analysis

and no exceptions to that procedure were noted.

Atomic Absorption Spectrometers

There is no evidence that any one of the different instruments used (see table 3) was superior.

The most consistent laboratories throughout (Labs A,

B, C) , in fact, each used a different type and/or

model. However, as shall be noted, the operating

environment, stability, and reproducibility of the

instruments were extremely important. Operating con- ditions varied, in many parameters, over wide ranges

and no discernible effects on overall accuracy are noted

.

Burner

This was a constant factor. Every laboratory

used the Boling type triple-slot burnerhead, and no difficulties were noted except during the course

of Exercises 3 and 4 when particulate residue

matter from the samples tended to clog the aspirator. Lab D replaced its burner for Exercise 5 and

then noted that its calibration curve became essen- tially linear, a condition not previously obtained.

Drafty ambient conditions affecting burner performance

undoubtedly played some role in the troubles encountered by Lab F

.

Oxidant-Fuel

All laboratories used air-acetylene for the oxidant-fuel mixture. The condition of the flame would

seem to be not critical. Lab D used an oxidizing

flame, the others either a slightly reducing or stoichiometric condition. It is recommended that

either of the latter two be used.

Pressure and flow conditions varied over quite

a wide range as shown in Table 4. No comments concerning flame conditions or rate of flow were received

83
with one exception. Lab A said it was important not

to vary or to allow variations in flame conditions

during the course of a run and recommends a welldesigned and stable gas handling system.

Stability and Reproducibility of Readout

By this is not meant solely the stability of the

readout device alone (usually digital) , but the sta- bility and precision of the entire instrumental system as recorded at the output. Through Exercise 3,

variations in readout to + 1% were tolerated, but

after the June 1971 meeting, all participants agreed

that reproducibility at the +0.5% level was necessary,

if the overall accuracy goal of + 1.0% of the true value was to be made achievable. In Exercise 4 and

5, Lab F dropped out of the network when it was evident that the instrument could not meet this specification. From the start, Lab F had difficulties

in this regard (see Comments Section) . Lab E aborted

Exercise 4 for the same reason, although sampling

difficulties probably contributed to its difficulties

.

Upon reloaction of the instrument, with more stable

electrical line conditions, Lab E rejoined the network

for Exercise 5. Lab C noted in Exercise 1 that its noise level was high, but made no further comments on the matter

and met the precision requirements throughout all

five exercises. Lab K noted an improvement in noise

levels after optical surfaces were cleaned.

Linearity

No special studies were performed to ascertain

the linearity of the calibration curves , although

comments on non-linearity were received by Labs D and F (see Comments) . The procedure calls for

bracketing the unknown solutions with the standard

solutions whose calcium values are close to that

84
of the unknown. Linear extrapolation is used and

small departures from linearity are disregarded,

although each laboratory ascertained the overall

linearity of its calibration curve. Effects of

non-linearity in the range for which this method

is applicable are small compared to other sources

of bias,

e. Source

All laboratories used calcium single-element

hollow- cathode lamps. Current conditions ranged

from 7-15 mA (Table 3) . No comments concerning

this parameter were received and none were made

during the June 19 71 meeting. Evidently, the source

and source conditions are not a factor affecting

accuracy at the level of this method.

It should be emphasized that all participants in

the work were completely familiar with atomic absorption spectrometers, could and would recognize instrumental difficulties, and, in some instances,

could make the necessary adjustments without help

from service personnel. It was also apparent that

the instruments—by and large--were kept in optimum

operating condition, although Labs E and F had

special difficulties that however reflect in no way

on the expertise of those investigators. It is safe to conclude that a properly maintained spectrometer of the 3 types used (it is not possible to speculate on other instruments) is capable of reproducibility, over several hours, of +0.5% and is

certainly not the limiting factor in the accuracy of

this method.

Reagents

Reagents and their quality are specified in the method,

all instances, standard solutions were prepared using

85
the NBS-CaCO^ (SRM 915) . In any referee method the use

of certified Standard Reference Materials is advisable as

a bias, in the form of unknown impurities, for example, will

distort the results in an unknown way. This SRM, supplied

and certified by NBS , is of very high purity and although

only certified at 99.9+% purity, it is known that the purity,

in fact, is at least 99.99%. Furthermore, metallic impurities

are known to be small and no significant error is introduced

into the system when SRM 915 is used. One word of caution is

in order. The SRM is apt to take up a small amount of moisture rather quickly, especially on warm, humid days. Weighing of the material should be accomplished expeditiously.

One investigator (Lab A) recommended that 10 times the amount

of material specified in the method be used in making up the

standard solutions and that then an additional 10-fold dilution be made. If quick weighing is done, this additional

precaution is not considered necessary.

Clinical laboratories very often do not have adequate

supplies of high-purity water. It is highly recommended

that both deionized and deionized distilled water be available. All laboratories used as a minimum deionized water,

but two labs (Lab A and C) had deionized distilled water

also available. Proper rinsing of all glassware requires

copious amounts of pure water and serious error can result

if the calcium contained in detergents and tap water is not completely removed during rinsing.

La

2 03

, available from at least the 3 sources used in

this work, was known to contain less than 15 micrograms

calcium per gram of the oxide. It is not available as an ACS Analytical Reagent (AR) Grade chemical, and all due

precautions should be taken to assure the purity of the

La^O^ used.

All other reagents used were ACS-AR Grade and are readily available from a number of reputable manufacturers.

86
C . Glassware

All glassware used in the sampling or dilution processes must meet the NBS Class A specifications. This

involves some additional expense, but is required if errors from this source are to be held to less than 0.1%.

The stratagem of using the identical 10 ml volumetric

pipet throughout for sample, blank, diluent, and standard

solutions preparation removes the possibility of variable

errors in these steps. There is no question but that the

time required to run the procedure is markedly increased

by this requirement because of the care that must be

taken in assuring no cross-contamination between the

various solutions. However, until shown (which is now possible) that errors introduced in using several pipets are small, this requirement must be considered an integral part

of the method. In one instance, an entire exercise had to

be rerun (Lab B - Exercise 5) because the operator deviated

from this requirement and used three different pipets. other

variables were also introduced simultaneously and it is not

possible to say what contribution to the 1-2% high results

of the discarded exercise were due solely to the variation

in pipet specification.

Proper cleaning of glassware is of critical importance.

The recommended procedure for cleaning all glassware is taken

from the candidate method of Pybus , et al . , and is the minimum

required. ^ab C used a more rigorous procedure, which is

described in the Comments Section. The final rinsing in

deionized distilled water is highly recommended and is standard practice at NBS when ultra-trace level work is performed.

In one instance an entire exercise had to be rerun (Lab

H - Exercise 5) when the first results reported were found

to be extremely variable. (Deviations from the ID-MS values

ranged from - 2.5% to + 6.4%, well outside the precision

previously experienced for that laboratory). Upon a close

examination of all steps in the procedure, a white residue

87
was found on several volumetric flasks and it was also found

that the rigorous rinsing steps prescribed had not been followed in detail. A rerun of the exercise brought the results

shown in section VII.

D . Aliquot and Dilution Steps

In the first exercise, several labs used a 1 ml pipet

and a 1:50 dilution--an option allowed in the candidate

method as published. In fact, Lab C used an entirely different dilution procedure, which was their standard practice.

For Exercise 2 and in the subsequent exercises , these

options were removed and the one 10-ml pipet technique

required, and all pipetting and dilutions were performed

as required with two exceptions. one exception (Lab B - Exercise 5 is described above. The only other exception

(Lab C - Exercise 3) came about when it was found that

insufficient sample did not allow rinsings to be performed

in the manner being practiced in that laboratory at that time.

The protocol used for Exercise 3 was not specific enough in

spelling out the rinse procedures and this was corrected in

the revised protocol used in Exercises 4 and 5. Three laboratories called into question the pipet rinsing procedures CLab A, C, and D) and this step was made more

specific in the final protocol used in Exercises 4 and 5. Two minor exceptions to the rinse procedures are noted: Lab A used a HCl-HNO^ (.3:1) rinse to improve the drainage of the

pipet and Lab F in Exercise 1 used a E^O rinse instead of

diluent. Apparently these deviations were unimportant in

introducing any significant bias

.

In only one instance was temperature allowed to vary in an uncontrolled manner CLab B - aborted Exercise 5). All

laboratories were instructed to allow solutions to reach room temperature before final dilution to the mark. Jo ex- ceptions or difficulties were noted in this regard.

88
' E. Sample

As mentioned in section IV-A, the scope of this

| method does not include sampling procedures. However,

certain observations may be made based on this work. The

pooled sera for Exercises 3 and 4, when finally received by

j the cooperating laboratories, were in quite a sorry state.

! Debris and bacterial growth were plainly evident. In fact,

| Lab D commented, "We would not have accepted this sample for

j analysis if it had been presented in our routine workload.

I Yet, overall it will be seen the results were not biased

' that strongly. It is felt that pipetting errors on these two

j samples accounted for most of the bias shown in the results.

Furthermore, the ID-MS staff had equal difficulties in their

aliquoting as shown by decreased precision in their results.

Lab A commented, for example, on Exercise 4 that "precision

ranged from 0.3 - 1.7% over 6 days on the Exercise 4 samples."

Precision on Exercises 1 and 2 were, as shown, much better

and more consistent. Pipetting difficulties were also com- mented on by Lab C for both Exercises 3 and 4. In the opinion of Lab E, the sample was so bad that Exercise 4 was

I not run.

It may be asked why the Experts Committee allowed these

samples to be run. A conscious decision was made to use these two "bad" sera samples in order to determine how far

afield the results would scatter. We may conclude that quite

murky, turbid solutions may be handled by the method as long

as actual precipitates, debris, or growth are absent.

F . Instrument Reading and Valid Measurement Criteria

In Exercises 1, 2, and 3, the concept of a valid measurement was not incorporated in the protocol. During the discussions of all the participants during the June 1971 meetj ing this became a major topic. Because there is inherent

in the atomic absorption spectrometer the possibility of

fairly short time instabilities or drifts (electronics,

flame, aspirating variations, etc.) during the interval

89
between readings, it was agreed to set up criteria for

accepting or rejecting data. These criteria are set forth in the final method and have the effect of limiting

the instrumental errors to + 0.5%. As many of these

instabilities are essentially random in nature, the

requirement that the reported result be based on the mean

of ten readings reduces this error even further.

Although the laboratories were requested to supply the

number of sets of data rejected, only Lab C provided this

information and then only for Exercise 4 (see Comments

Section) . It is suspected that some of the difficulties may

have been due to the poor nature of the sample which may

have caused aspirator clogging. It would have been extremely

interesting to have had this information for Exercise 5. With the one exception of Lab D in Exercise 4, all laboratories used the ten replicate reading for Exercises 4 and 5. After Exercise 3 at least one laboratory (Lab C) did not feel that the protocol was clear or definitive enough on defining the run order sequence of standards and samples (for more than one unknown sample as was the case throughout)

.

This was clarified in the final protocol.

G . Deviations from the Pybus , et al . , Method

As explained earlier, the candidate method was considered

to have been well-thought out enough and studied so that ex- tensive investigation in this study was not necessary. Several

options do exist in the candidate method. Some of these have now been removed, others remain because no effect could be at- tributed one way or the other. Options in the original method

now removed include: ten replicate reading requirement ; 10 : 500

dilution factor; and use of one 10 ml pipet. Exceptions made

during the course of this work have already been discussed.

Options remaining are principally the use of Sr as an internal reference, and the addition of a Mg standard (very

often in clinical use Ca and Mg are determined together)

.

90
[ Laboratories B, H, and K used the Sr internal reference

option throughout, the remaining labs did not. There is no evidence from the results that an internal standard improves

I the accuracy of the method.

Magnesium standard was used by Labs B and C, the others

|| did not. No major effect can be attributed to this factor.

Lab B showed in Exercise 1, as the result of some addi-

! tional work, that the addition of Na and K was necessary in all

1 solutions —blank, diluent, standards, and unknowns. Exercise

| 1 samples did not contain Na and K; (it was thought to test the

! labs for precision primarily on the simplest possible solutions)

.

| Lab B commented that if the standards contained Na and K while

the test samples did not then results should be about 1% high

from the true value. As can be seen for the results from Ex- ercise 1, this conclusion is not borne out. However, it was never ascertained how many of the laboratories added Na and K to the test samples as this point was well known to them. In any case, this question is rather academic because the final

method is designed to measure calcium in serum or solutions

containing physiological amounts and proportions of Na and K. Significant deviations from the procedure other than those

already noted include:

1. A substantially different calibration procedure (Lab

D - Exercise 1) . Lab D followed the protocol exactly

in Exercise 5. 2. No blank solution used (Lab D - Exercise 1). The ar- gument may be made that an automatic blank correction

is made if measurements are not made on an absolute

basis, but relative to the standards as both solutions

contain the same chemicals and diluent. This may be

true, but the value of measuring the blank is to as- certain the magnitude of the calcium blank that should

be low relative to the concentrations being sought as blanks are notoriously variable in nature and may re- sult in large biases (often random and unknowable)

.

91
3. In the comments and suggestions accompanying the

first protocol, the point was strongly made that

a sufficient block of time apart from other responsibilities should be made available to the analyst

during the running of each exercise. Only Labs A

,

B, and C were able to or did do this consistently

throughout. The other labs are all operating clinicai

laboratories and this work was scheduled to fit into

the routine work load. It is difficult to assess the ! overall contribution to accuracy of this factor.

It will be noted, however, that these three laboratories performed most consistently throughout these

studies

.

4. One minor point for the record—Lab E in Exercise 1 used a procedure based on another method, but then

followed the prescribed protocol thereafter.

H. Discussion of General Points

It would be a mistake to assume that this study was accomplished under ideal conditions and that all parts of

the network were under strict control at all times in all

exercises . A careful reading of the Comments Section shows

how many unplanned variables actually entered the system.

Only in Exercise 5 did the network operate in what could be

considered adequate control. In the two instances in Ex- ercise 5 where significant deviations were introduced (Lab B and H) , quick recognition of these deviations was made

by those laboratories and complete reruns were made according

to the protocol. Many initial departures were made because

there was not a clear understanding in the minds of all the

cooperating investigators as to what was allowable and what

the underlying philosophy of operation was. It was only

after the June 1971 meeting that the point of "no deviation

from the protocol" was fully accepted. This study, therefore,

should be considered in no small part to be educational in

92
nature. Scientists in clinical chemistry need to be made

aware of and then to accept the importance of properly developed referee methods if a meaningful measurement system

is to be realized in practice. It is hoped that the present

study will serve as a model for the development of much

needed additional referee methods, recognizing the many

areas where mistakes in this study were made. Some recom- mendations to bring about more ideal conditions for future

studies are given in section X.

It is readily apparent that the validity of this work

rests on the thesis that the isotope-dilution mass-spec-

:| trometry reference-line method, against which all the data

| was referred, is, in fact, a method of proven accuracy. The

data and evidence in section VII strongly support this thesis

In fact, the Experts Committee purposely chose calcium as the first referee method to be developed because of the very

I strong competence of NBS in this area. The question

naturally arises —how is a referee method developed when

such a base-line reference method is not available? There

is no doubt but that the ferreting out of systematic biases

then becomes a much more complex and time-consuming task.

In such instances , the preliminary studies and research that

went into the candidate method must be of the highest caliber

interferences studied, specificity and purity of reagents

evaluated, instrumental parameters and errors elucidated,

etc. These questions should be fairly well-settled before

the method is to be considered a candidate for the development of the referee method. It is evident from this work

that only a relatively few variables can be handled within

any reasonable expenditure of time, effort, and money.

Screening and evaluation of candidate methods by the Experts

Committee are thus seen to be of critical importance for

future work. When an independent reference- line method is

not available, then consensus among experts, but based on

93
studies done under controlled conditions, is the only viable

alternative to the methodology used herein. In this regard,

the professional societies, through their standards committees

and experts panels, can and should play a leading role.

The question may be raised, "If a reference-line method is already available for use, why not make it the referee

method?" In principle there is no reason why this could

not be so, but in practice, the reference- line method may

have limitations that preclude its adoption as the referee

method. This is certainly the case in this instance. There

are few, if any, clinical laboratories in the world that

have either the equipment or trained personnel to use the

ID-MS method described herein; nor would it be practical

to suggest its incorporation into the clinical milieu.

Unfortunately, it is often true that the most accurately

based analytical systems are also those most expensive,

Doth in equipment and personnel.

Although the documented evidence in this study will

not rigorously bear out in all instances the following

conclusions, there is little doubt of the validity of

these assertions:

1. The development of a referee method, given a well-studied candidate method, is essentially an exercise in analytical , not clinical chemistry.

The quality, training, background and motivation

of the participants, especially those who do the

actual laboratory work, must be of the highest

caliber. The educational implications for clinical

chemistry are obvious. It probably is not fair to ask fully committed and very busy technologists to participate during the course of the work. Motivation

will probably be lacking, unless they were involved in

the studies from the start and feel they have a personal stakes in this work.

94
Time must be made available for the participating

scientist. Referee development work cannot be con- sidered an adjunct or auxiliary responsibility.

Again this requirement militates against using

cooperating laboratories that are fully committed

to high-load daily routines unless no other alternative is available. It should not be concluded

that because several of the laboratories in this

work are "working" laboratories, that their results were affected adversely because of this pressure.

These laboratories produced the results they did

in spite of the daily work load. It would not be

fair to ask these laboratories to continue on a voluntary basis into the indefinite future. Resources

must be made available so personnel dedicated solely

to future work can be freed of routine responsibilities —in other words, research in these laboratories

should be independently supported.

Funding in support of the cooperating laboratories for

future work is essential. Only when the work is done

in a business like, contractual mode can the necessary

controls be instituted and maintained. In this work,

the question of controls and direction is academic.

All laboratories gave freely of their time and accepted direction in a truly remarkable cooperative manner. There were no prima donnas, no quibbling over details,

only valid criticism and valuable suggestions. In the more complex studies to follow, these conditions may

not apply, and then it will be important to have the

central control that is only possible when the work

is funded under a contractual arrangement.

95
IX. CONCLUSIONS

A referee method for the determination of calcium in serum and/or solutions containing physiological amounts of

sodium and potassium has been tested and validated. When

the protocol given in section IV is followed in every

detail, subject also to the many provisos and conditions

listed in the Discussion Section, then this referee method

will give the concentration of calcium accurate to within

±2% of the "true" or absolute value in the range from 3 to

6 milliequivalents calcium per liter of sample.

Inherent in the referee method are the conditions and

findings of the basic candidate method of Pybus , et al. , upon

which this work was based. This is especially true for

interferences and matrix effects that were not studied in

this work. Significant departures from the method with

regard to these factors may lead to substantial systematic

errors . Both human and bovine pooled serum and aqueous

solutions containing physiological amounts of sodium and

potassium may be analyzed with assurance by the referee

method. The effect on accuracy of scaling down the sample

size or using a less than 10:500 dilution has not been

tested although some additional work (not reported herein)

indicates that smaller sample sizes and lesser dilution

factors might be tolerated.

It is certain that the spectrometer used must be in

capable, knowledgeable hands, in compatible surroundings,

and in tip-top operating condition, especially with regard

to stability and reproducibility. Furthermore, no deviation

from the measurement reading procedure can be tolerated,

as the error from this source alone is likely to approach

+_0.5%.

Finally, the evidence strongly supports the contentions

made in the Discussions Section that the method must be developed by scientists well trained in basic analytical

96
chemistry techniques and who are, in addition, meticulous

throughout and highly motivated.

Note Added

At the conclusion of Exercise 5, it was thought by the

Experts Committee to be an interesting experiment to send

samples of the same serum utilized in Exercise 5 to several

commercial and hospital laboratories

.

The results of this small survey are given in Appendix 6

.

Because of the limited laboratory sample size and the lack of

rigorous control, we recommend that no far-reaching conclusions

be made based on this work alone. However, the results are illuminating.

It should be noted that one laboratory, Lab R, produced

results consistent with the known values. Here, there is the

possibility of examining its procedures to see if a faster,

less time-consuming referee method can be developed.

97
X. RECOMMENDATIONS FOR FUTURE REFEREE METHOD DEVELOPMENT

STUDIES

To the best knowledge of all concerned in this work, this

study is the first made to bring about in a systematic way a referee method in the field of clinical chemistry. It is

quite natural that, ex post facto, one now sees the many weaknesses, unnecessary work, inconsistencies, false starts, etc.,

that might have been avoided through better planning, tighter

scheduling, more explicit directions, and closer coordination.

In a real sense this study should be considered to have been

part of an educational process from which future work will

surely benefit. Taking advantage then of hindsight, several

recommendations are worth listing.

A. Preplanning Session

No future referee method development study should start

until all participants have agreed to meet in person at some agreed on location. Prior to this session, the Experts

Committee should have selected, after some considerable study,

the candidate method, have set the accuracy goal* and agreed

on the factors most likely to be the principal contributors

to the systematic errors. The Experts Committee should have

prior to this meeting also have met with the Coordinator

and statisticians to agree on the experimental design,

statistics, etc. In this study, the meeting of the participants did

not take place until after the third exercise. It was then that many details were agreed on that should have

been ironed out prior to any of the laboratory work

.

For example, it was only at that meeting that the "non- deviation from protocol" principle was really accepted.

B . Coordinator

The coordination of the work, gathering the data,

sending out the samples, establishing and maintaining

communications, and a hundred small details are in the

98
hands of the project Coordinator. In this work the

Coordinator was J. Paul Cali whose primary duty at NBS

is the management of the Standard Reference Materials

program. Most of the inefficiency, delays in scheduling,

etc. , can be attributed to the fact that the Coordinator

role was secondary to his primary responsibilities, and

consequently often the coordinating duties had to take

second place. As this work is of the highest importance

to the Nation, a full-time Coordinator is highly

recommended for future work, especially if the tempo

of this work is to be stepped up and increased—both

highly desirable goals in view of the vast amount of

referee development work to be done.

If additional financial support is made available

(next paragraph) , then the Coordinator could also act

in the role of project manager for: (1) Establishing

and monitoring contractual arrangements; (2) Evaluating

proposals, and (3) Visiting proposed cooperating laboratories to establish availability of qualified personnel,

equipment, etc. These additional tasks would certainly

warrant a full-time Coordinator.

C . Financial Support

Without going into great detail, the true cost of

developing this referee method was, conservatively,

$120,000. Of this amount, probably $40-50,000 of free

service was contributed jointly by the Experts Committee

and cooperating laboratories. Approximately $40,00 0 was contributed by NBS (again free to the project) in the

salaries and overheads of the Coordinator, his staff,

and MBS statistical services. Only $40,000 in direct

dollar appropriations was ever charged to this work

at NBS, primarily for the ID-MS work.

It is highly recommended that future referee development studies be fully funded, especially in support of

99
the cooperating laboratories. If this support can be

made available, it will accomplish several important

ends. It will allow: Fully-qualified and well-trained

scientists to be recruited and paid to participate,

as opposed to the present situation where scientists must

be taken away from other pressing duties; (2) Under

contractual agreements, greater control, flexibility,

and scope of the work —necessary reruns, more exercises, more comprehensive study of the variables, non-interference

by routine work, etc.; (3) Tighter and more intensive

scheduling—more work in a shorter duration; and (4) Purchase of necessary supplies, updating or renovating of new equipment.

D . Experts Committee

If financial support can be found, then it is reasonable to expect a more concentrated, extensive study to

be made by a paid Experts Committee, rather than one that

works completely on a voluntary basis. In this work, it was fortunate that a very well-studied candidate method

was already available. In the future this may not be

the case, and then the Experts Committee might have to conduct, or have done under their guidance, laboratory work

to establish the adequacy of the candidate method.

As has been explained earlier, the availability of

an independent reference line method against which the

candidate method can be tested is most desirable. An

Experts Committee who could spend a fair amount of time

on this work, and that implies financial support, could

encourage and support developments in this area. For

example, studies of isotope dilution techniques using

radio-tagged cholesterol or bilirubin would seem to be

a subject worthy of support and might provide the independent reference- line method that is not now available

for these classes of clinically important substances

.

100
An undertaking of this magnitude involves many people,

and, in this instance, many different organizations. It is not possible to give personal credit in every instance be- cause many are not known to the author by name. Valuable

advice and expert guidance were received from the Experts

Committee—Drs . George N. Bowers, Jr., Donald S. Young, and

Nathan Radin. Suggestions concerning some overall philosophy

were made by Dr. Russell J. Eilers.

Our deepest thanks are due the cooperating laboratories

and the principal investigators whose names and affiliations

are given in Section II-A. Without their whole hearted

cooperation and enthusiam this work would not have been

possible at all. At the same time we acknowledge the con- tributions of all the scientists and technologists at these

laboratories who participated in the actual work.

Special thanks is given to Hartford Hospital (Dr. George

N. Bowers, Jr.) and to the Center for Disease Control (Dr.

Joseph H. Boutwell, Dr. David D. Bayse and Ms. Dorothy S. Lewis) for the preparation and contribution of the pooled

sera used in Exercises 3, 4, and 5. At NBS many scientists participated in the work: (1) For the isotope-dilution mass-spectrometry reference line

studies under the direction of William R. Shields, we thank

Ernest L. Garner (mass spectrometry) and Keith M. Sappenfield,

Larry A. Machlan, Thomas J. Murphy, and John R. Moody (isotope

dilution, preparation of unknown solutions). (2) For

reduction of data and assistance with the statistical analysis

thanks is due Miss M. Nancy Steel. All the isotope-dilution mass- spectrometry work and the NBS participation as one of the

cooperating laboratories was performed in the Analytical

Chemistry Division. The Chief of that Division, Dr.

W. Wayne Meinke, wholeheartedly supported this effort and

always gave this work the highest priority, even in these

101
times when competition for scarce resources is great. We

acknowledge and thank him for this support.

The staff of the Office of Standard Reference Materials

helped in many ways during the course of the work

:

preparation of the data recording sheets , correspondence

with the cooperating laboratories, mailing of the samples,

etc. Special thanks is due Mrs. Suzanne C. Love for keeping

many details straight, and for filing of the incoming data.

For the typing of the manuscript thanks are due to Mrs. S. Love and Mrs. V. Davis.

102
REFERENCES

[1] Eisenhart, C, Realist Evaluation of the Precision

and Accuracy of Instrument Calibration Systems,

J. Res. Nat. Bur. Stand. (U.S.), 67C, No. 2,

161-187 (Mar. -Apr. 1963)

[2] Cali , J. P., Accuracy in Clinical Analytical Methods

and Its Attainment (In publication)

[3] NBS Clinical Laboratory Standards, SRM Brochure (1971)

Office of Standard Reference Materials, Washington,

D.C. 20234

[4] Pybus , J., Feldman, F. J. and Bowers, G. N. Jr.,

Measurement of Total Calcium in Serum by Atomic

Absorption Spectrophotometry, with Use of a Strontium

Internal Reference, Clin. Chem 16, 998-1007 (1970)

[5] Bowers, G. N . , Jr., et al, The Preparation and Use of

Laboratory Control Materials for Monitoring Precision

in the Clinical Laboratory, Standard Methods of Clinical Chemistry, Vol. 7 (in preparation).

[6] Trudeau, D. L., and Freier, E. F., Determination of

Calcium in Urine and Serum by Atomic Absorption

Spectrophotometry, Clin. Chem. 13_, 101 (196 7) 17] Shields, W. R. , Editor, Nat. Bur. Stand. (U.S.),

Tech. Note (in preparation)

.

[8] Garner, E. L., Machlan, L. A. and Shields, W. R. , Nat. Bur. Stand. (U.S.), Spec. Publ. 260-27, 150 pages,

U. S. Government Printing Office, Washington, D.C,

20402 (1971)

.

19] Catanzaro, E. J. , Champion, C. E. , Garner, E. L.

,

Marinenko, G., Sappenfield, K. M. and Shields, W. R.

,

Nat. Bur. Stand. (U.S.), Spec. Publ. 260-17, 70 pages

U.S. Government Printing Office, Washington, D.C.

20402 (1970)

.

110] Catazaro, E. J., Murphy, T. J., Garner, E. L. and

Shields, W. R., J. Res. Nat. Bur. Stand. (U.S.),

7 OA (Phys. and Chem.) 6, 453-458 (Nov. -Dec. 1966).

Til] Shields, W. R.^ Murphy, T. J. Garner, E. L. and

Dibeler, V. H., J. Am. Chem. Soc, 8^4, 1519-1522

(1962) .

103
112] Shields, W. R., Murphy, T . J. Catanzaro, E. J. and

Garner, E. L., J. Res. Nat. Bur. Stand. (U.S.), 7 OA

(Phys. and Chem.) 2, 193-197 (Mar. -Apr. 1966)

113] Shields, W. R., Murphy, T. J. and Garner, E. L., J. Res. Nat. Bur. Stand. (U.S.), 68A (Phys. and Chem.)

6, 589-592 (Nov. -Dec. 1964).

114] Catanzaro, E. J., Murphy, T. J., Garner, E. L. and

Shields, W. R. , J. Res. Nat. Bur. Stand. (U.S.),

68A (Phys. and Chem.) 6, 593-599 (Nov. -Dec. 1964).

115] Catanzaro, E . J., Murphy, T. J., Garner, E. L. and

Shields, W. R., J. Res. Nat. Bur. Stand. (U.S.),

73A (Phys. and Chem.) 5, 511-516 (Sept. -Oct. 1969).

116] Shields, W. R. , Garner, E. L. and Dibeler, V. H. , Jr. Res. Nat. Bur. Stand. (U.S.), 66A, (Phys. and Chem.)

1, 1-3 (Jan. -Feb. 1962)

.

117] Catanzaro, E. J., Murphy, T. J., Shields, W. R. and

Garner, E. L., Res. Nat. Bur. Stand. (U.S.), 72A

(Phys. and Chem.) 3, 261-267. (May-June 1968).

I18J Shields, W. R., Editor, Nat. Bur. Stand. (U.S.), Tech.

Note 277, 99 pages, U.S. Government Printing Office,

Washington, D.C. 20402 (1966)

.

119] Shields, W. R. Editor, Nat. Bur. Stand. (U.S.), Tech.

Note 426, 53 pages, U.S. Government Printing Office,

Washington, D.C. 20402 (1967).

104
((APPENDIX 1. NBS STANDARD REFERENCE MATERIALS

and REFEREE METHODS DEVELOPMENT

A Referee Method may be defined as a measurement method

(or technique designed to determine one or more characteristics

i or properties of a substance to a well-defined and achievable

• accuracy for a specific or well-defined use. In order to

j achieve the desired accuracy the method or technique must be

| capable of study to determine the systematic errors or biases

inherent in all measurement systems. For many systems this

study is most expeditiously and effectively carried out when

j a Standard Reference Material (SRM) is available.

j An SRM is by definition a well-characterized material

I whose certified properties are accurately known within some

| stated limit (s). Through the use of the Standard Reference

j Material, systematic errors in the method under development

may be discovered and eliminated, or if the latter is not

possible, then corrected. If this process is carried out in

several well-qualified laboratories, by a number of scientists

working independently—except for the common use of the identical SRM and identical specimens —then the emergence of a Referee Method becomes possible. !<vhen many workers obtain

equivalent accuracy with the method, then by consensus among

qualified experts we may say that a Referee Method has been

achieved.

Throughout this process one sees the central role of

the SRM. It is through the mechanism of the SRM that a useful transfer of technology occurs. ^BS by its very mission

and charter has been charged with this transfer of technology

and because it is the primary source of SRM's in the nation,

the Bureau can play a significant role in the aforementioned

process. NBS, through its Institute for Materials Research,

105
and its Office of Standard Reference Materials is now ready

to implement this process where needs are urgent, identifiable,

and in the national interest.

Several ingredients are necessary if this process is to be successful:

1. The undertaking must have the interest and active

backing of national , and in some cases international, professional , industrial, or standardizing

societies, groups or bodies. The participation of

these bodies in defining the problem to be studied,

deciding on the experimental parameters, guiding

the cooperating laboratories , and evaluating the

results should be delegated to a small (3-7) group

of well-qualified experts, drawn from the interested

societies and groups

.

2. The achievement of accuracy in the determination

of the characteristics or properties of important

classes of substances must be the overriding goal,

and is best reached through the development of a referee method based based on an SRM.

3. All participating elements in the referee process

must be in quality control at all times. This

implies tight statistical control and well-designed

and executed experimental techniques.

4. The dedicated commitment of a number of well-qualified

laboratories in support of the "round-robin" development and testing procedures is paramount.

5. When the accuracy goals of the method have been

achieved, NBS will publish the method as a "Referee

106
Method" in the publication series reserved for

Standard Reference Materials (NBS Special Publication

Series 260) . Simultaneous publication in professional

society journals, other government publications, or standards writing organs is not precluded, and indeed,

will be encouraged. These methods as developed will

also be referenced in the SRM catalog alongside the

pertinent SRM. NBS participation in this process, when all the necessary

conditions listed above have been assured, will consist of all or some of the following (as appropriate)

:

1. Provide the appropriate SRM to participating laboratories .

2. Provide the statistical design and experimental con- trols for the various stages of the experimental

phases of the work.

3. Provide, where technically feasible, samples of known

composition whose properties have been determined at NBS by an independent method of previously ascertained

accuracy. (NOTE: In many, indeed in most, cases an accurate independent method is probably not available

or if so, too complicated, complex, or expensive to be

considered as the candidate referee method, itself)

.

4. Collect, collate, and statistically evaluate the

cooperating laboratory results and report these to the experts team.

5. Participate as one of the cooperating laboratories.

6. Coordinate the activities of the cooperating laboratories, internal NBS work, and the experts team.

107
7. Publish the results and reference methods in the

NBS 260 Special Publication Series.

J. Paul Cali , Chief

Office of Standard Reference Materials

National Bureau of Standards

Washington, D.C. 20234

108
APPENDIX 2

U. S. DepartA

Maurici

f

of Commerce

. Stans

National I A. V. Standards

-Director Qkrttftrate nf Analgirijs

Standard Reference Material 915 Calcium Carbonate

This Standard Reference Material is certified for use in the calibration and standardization of procedures employed for the determination of calcium in clinical analysis. The sample consists of highly purified calcium carbonate, and chemical assay as well as

analysis for specific impurities indicate that the material may be considered to be essentially pure. Purity 99.9+ percent Water 0.01 ± 0.005 percent

Replicate samples taken from a randomly selected region of the undried material were assayed by a coulometric acidimetric procedure. The results from nine independent

determinations, based on expression of the assay as calcium carbonate, indicate a purity

of 99.99+ percent with a standard deviation of 0.003 percent. Samples equilibrated at a

relative humidity of 90 percent and assayed by this coulometric procedure showed a maximum moisture adsorption of 0.02 percent as compared to samples that were dried for 6 hours at 210 °C. The moisture content, similarly measured, on samples equilibrated

at 75 percent relative humidity, was found to be 0.01 percent. The water content was de- termined by the Karl Fischer method. The calcium carbonate used for this Standard Reference Material was obtained from

the J. T. Baker Chemical Company, of Phillipsburg, New Jersey. Analyses were performed by C. E. Champion, E. R. Deardorff, G. Marinenko, O. Menis, T. C. Rains, T. A. Rush, W. P. Schmidt, B. F. Scribner, V. C. Stewart, J. K. Taylor, and D. W. Vomhof

.

The overall direction and coordination of technical measurements leading to the certification were under the chairmanship of R. Schaffer. The technical and support aspects concerning the preparation, certification, and

issuance of this Standard Reference Material were coordinated through the Office of Standard Reference Materials by J. L. Hague.

Washington, D. C. 20234 W. Wayne Meinke, Chief March 4, 1969 Office of Standard Reference Materials

10 9
APPENDIX 3 - NAMES AND ADDRESSED OF PRINICIPAL INVESTIGATORS

AND COOPERATING LABORATORIES

1. Dr. George N. Bowers, Jr. ! Co-Director

Clinical Laboratory

Hartford Hospital

Hartford, Connecticut 06115

2. Dr. S. Raymond Gambino

Professor of Pathology and Director

of Clinical Chemistry Laboratories

College of Physicians and Surgeons

of Columbia University

Department of Pathology

630 West 16 8th Street

New York, New York 100 32

3. Dr. Alan Mather

Chief

Clinical Chemistry Section

National Center for Disease Control

1600 Clifton Road, NE Atlanta, Georgia 30333

4. Dr. John C. J. Porter

Biochemists and Chief

Department of Clinical Biochemistry

Toronto General Hospital

Toronto 2 , Ontario Canada

5. Mr. Theodore C. Rains

Research Chemist

Analytical Chemistry Division

National Bureau of Standards

Chemistry Building, Room B226

Washington, D. C. 20234

6. Dr. Royden N. Rand

Assistant Chief

Chemistry Division

Hospital of the University

of Pennsylvania

William Pepper Laboratory

3400 Spruce Street

Philadelphia, Pennsylvania 19104

110
7. Dr. Leonard Sideman

Chief, Biochemistry Section

Department of Health

Division of Laboratories

2100 West Girard Avenue

Philadelphia, Pennsylvania 19130

8. Dr. Donald S. Young

Chief, Clinical Chemistry

Department of Health, Education,

and Welfare

Public Health Service

National Institute of Health

Building 10, Room 4N 309

Bethesda, Maryland 20014

111
APPENDIX 4 . Preparation of Serum Calcium Control for

the National Bureau of Standards

Calcium Standardization Program

by

David Bayse, Ph.D.

Dorothy Sue Lewis, B.S. This calcium control was formulated as follows

:

1. Three liters of bovine serum were dialyzed against

saline for 24 hours. The saline was changed twice

during this period.

2. This dialyzed serum was assayed by calcium atomic

absorption and was found to contain 1.39 meq/1

calcium.

3. This dialyzed bovine serum was mixed with undialyzed

bovine serum with a calcium analysis of 4.69 meq/1.

x^ight liters of such a mixture were prepared with a final calcium concentration of 3.6 meq/1. This

pool constituted both the low pool and the basic

matrix for concentration of this study. Antibiotics were added to this matrix:

672 mg of streptomycin, USP

1,600,000 units of penicillin, USP

20 mg of fungizone, USP

4. To the 2.9 3 liters of the low calcium pool were added

23.2 mis of calcium stock solution (402.2 meq/1

calcium) . This solution was allowed to equilibrate

for 24 hours and was stack filtered using Millipore

filters: prefiltered - 1.5 microns, 0.8 microns,

0.6 microns, 0.45 microns, 0.3 microns, 0.22 microns.

The final concentration of this solution was 5.69

meq/1

.

112
5. The two intermediate pools were prepared from mixtures

of the high and low calcium pools using a Mettler P10

balance. One of the two intermediate pools consisted

of l/3rd of the high pool mixed with 2/3rds of the low

pool by weight, with a resulting calcium analysis of

4.29 meq/1. The second intermediate pool consisted

of 2/3rds of the high pool and l/3rd of the low pool

by weight, and the resulting calcium was 4.9 5 meq/1.

6. The material was then sterile filtered using a 0.22

micron Millipore filter and 50 mis were dispensed into 60 ml sterile Wheaton bottles. This material was tested for sterility by microbiological examination.

113
2 °

•H >

^3 g

114
APPENDIX 5 Sheet 2

Analysis

Exercise No. Run No. Lab Code

NBS Sample

# NBS Sample

# NBS Sample# NBS Sample

# Absorbance

Values Absorbance

Values AbsorbanceValues

Absorbance

Values

NBS Sample

# NBS Sample

# NBS Sample

# NBS Sample

Absorbance

Values

Absorbance

Values Absorbance

Values

Absorbance

Values

115
APPENDIX 5 Sheet 3

Exercise No. Run No. Method

Date received Date reported Instrument (Company) (Model #) (Serial #) Wavelength, ^ Slit width \i Source, (single or multielement) hollow-cathode lamp Current , mA

Burner , (type) Oxidant, flow rate 1/min. Fuel, flow rate 1/min.

If flow rate unknown give: Oxidant (PSI); fuel: (PSI) Flame condition, (oxidizing, reducing or stoichiometric)

Time, constant, _sec. Scale expansion X. Readout obtained with (recorder, digital or ) Standard stock solution, mg/1, prepared from . Calibration curve, working range to yg/ml. Laboratory Ambient temp °C to °C (over course of entire exercise) Interference Filter (If used) : type; nom. wavelength

Other comments: (Note especially deviations from Bowers method)

116
APPENDIX 6 - RESULTS FOR CALCIUM (USING EXERCISE 5 SERUM)

FROM 9 LABORATORIES USING ROUTINE METHODS.

(Donald S. Young)

The performance goals of this study were achieved with

the Reference Laboratories only after five rounds of analysis

of samples. These laboratories were selected in the first

place for their reputation for high quality work and the concern of their directors for accuracy and precision in analysis.

All of these laboratories used atomic absorption spectrophotometry

for measurement of calcium. We felt that it would be interesting to observe the performance of other laboratories without

the intensive preparation that was necessary to achieve the

satisfactory performance eventually accomplished by the Ref- erence Laboratories. We initially made the assumption that

atomic absorption was the best method for assaying calcium in

physiological fluids, without evaluation of other techniques.

Therefore we elected to send the same samples that were analyzed

in the fifth round by the Referee Laboratories to several

hospital and commercial laboratories for analysis by their

routine methods. The type of laboratory and technique used is

summarized in Sheet 1.

Each of the four serum samples analyzed by the Reference

Laboratories in the fifth round of the study was divided into

two parts (labeled 1 and 2) . These were sent as samples of

unknown concentration to each of the participating laboratories

.

The staffs of these laboratories were requested to analyze the

samples without special handling. None of the analysts was aware that the two samples labeled with the same code letter were aliquots of the same serum. The aliquots were prepared

in the Clinical Pathology Department, National Institutes

of Health. The aliquots were made from several bottles of

the serum used in the fifth round of the survey of the

117
Reference laboratories . The aliquots were dispensed into

acid-washed Vacutainers and stored at 4°C for less than one week before analysis. The aliquots were clear and appeared

to be homogeneous. However, the Director of laboratory P initially questioned the homogeneity of the samples. The

Director of laboratory X noted the presence of a slight deposit in the samples that he received. None of the other

laboratory Directors commented on the samples

.

The results of the analysis of the samples by the

different laboratories are presented in Sheet 2. Within

all laboratories for each technique there is good consistency

between the duplicate measurements. The largest discrepancy

observed was 0.2 meq/1. One laboratory (laboratory R)

found no difference between the paired results . However

in those laboratories in which two methods were used there

was poor agreement between the mean results by the two techniques. In laboratory P, although there was internal

consistency when the samples were analyzed at night and

during the day, the mean values differed. The laboratory

Director believes that the analyst at night failed to mix the

samples adequately prior to analysis.

For each sample there was a considerable spread of re- sults when the performance of all laboratories was compared.

Thus results for sample A varied from 3.8 to 4.7 meq/1,

for sample B from 3.25 to 4.3 meq/1, for sample C from 4.85

to 6.0 meq/1, and for sample D from 4.25 to 5.6 meq/1. The

spread of results in each case brackets or exceeds what would

normally be considered as the normal range of results of a healthy population. With the diagnosis of a parathyroid

tumor still determined to a large extent by the concentration

of calcium in a patient's serum several unnecessary and

dangerous operations would have been indicated, on the basis

118
of the results obtained by the different laboratories.

There was no consistency between the laboratories using

the same technique suggesting that factors other than methodology are responsible for the variation in results. That it

is possible for excellent results to be achieved under routine

conditions is indicated by the performance of laboratory R. All the duplicate values were identical and all agreed with

the isotope dilution values obtained at NBS and the mean

value of the Referee laboratories . While it can not be

guaranteed that no special precautions were taken for the

analysis of these samples in laboratory R it is gratifying

to note its performance because it is one of the biggest

commercial laboratories in the country. The impact of its

performance is probably greater than that of any other laboratory. On the other hand the performance of another large

commercial laboratory (U) was the worst of all the participating laboratories. As a consequence of this study this laboratory has now identified the cause of its poor performance and

corrected its deficiencies.

This study has confirmed the widely-held belief that

calcium measurements are generally inaccurate and lack

consistency from laboratory-to-laboratory. However, the

performance of some of the laboratories indicates that good

results can be achieved under normal operating conditions

.

This study confirms our contention that in order to achieve

meaningful values in clinical laboratories a national program

on a scale much broader than that envisaged hitherto, must

be undertaken to educate analysts and examine the cause of

the deficiencies in their laboratories

.

119
APPENDIX 6 Sheet 1 TYPE OF LABORATORY AND METHODOLOGY USED

LABORATORY TYPE TECHNIQUE

N Hospital Atomic Absorption

P Hospital EDTA Titration

Q Hospital EDTA Titration

R Commercial Atomic Absorption

S Commercial a) Colorime trie EDTA

(SMA 12/60)

b) Flame emission

T Commercial Fluorometric EDTA

(AutoAnalyzer)

U Commercial Atomic Absorption

W Commercial EDTA Titration

X Hospital a) EDTA Titration

b) Atomic Absorption

120
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121
I
FORM NBS-H4A (1.71) U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

PUBLICATION OR REPORT NO. NBS SP260-36

2. Gov't Accession No.

3. Recipient's Accession No.

4. TITLE AND SUBTITLE A Referee Method for the Determination of Calcium in Serum

5. Publication Da May 1972

6. Performing Organization Code

7. AUTHOR(S) J. P. Call, J. Mandel, L. Moore and D. S. Young

8. Performing Organization

9. PERFORMING ORGANIZATION NAME AND ADDRESS NATIONAL BUREAU OF STANDARDS DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

10. Project/Task/Work Unit No. 3020101

11. Contract/Grant No. N/A

12. Sponsoring Organization Name and Address Same as No. 9

13. Type of Report & Period CoveredFINAL

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summary of mo:

bibliography or literature survey, mention it here.) significant informati includes a significant

The first referee method of analysis (a method

of known accuracy) in clinical chemistry has been

developed by a team of experts from clinical chemistry,

8 cooperating clinical chemistry laboratories, and

scientists from the National Bureau of Standards.

Subject to the limitations and provisos stated, calcium

in serum can be determined by the referee method to within +2% of the "true or absolute" value. Also

discussed are: (1) What Constitutes a Meaningful Measurement System; (2) Theory of Operation and Practice Required to Develop a Referee Method; (3) History of the Actual Work; the Referee Method Protocol;

(4) Results; (5) The Isotope-Dilution Mass Spectrometry Work; (6) Discussion, Conclusions, and Recom- mendations for Future Referee Method Developments.

17, ^JcWScy^lp ^t6Mffdlbl^^^6ne^^rometry; calcium in serum; clinical chemistry; referee method for calcium.

18. availability statement

[XKl'nlimited. FOR OFFICIAL DISTRIBUTION. DO NOT RELEASE

TO NTIS.

19. SECURITY CLASS

(THIS REPORT)

UNCLASSIFIED

20. SECURITY CLASS

(THIS PAGE) UNCLASSIFIED

21. NO. OF PAGES

136

22. Pr$1.25

U. S. GOVEI1NMKNT PR INT INC. OFFICE : 1972

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