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CLIN. CHEM. 40/6, 934-938 (1994)
934 CLINICAL CHEMISTRY, Vol. 40, No. 6, 1994
New International Reference Preparation for Proteins in Human Serum (RPPHS)
John T. Whicher,1 Robert F. Ritchie,2 A. Myron Johnson,3 Siegfried Baudner,4 Jacques Bienvenu,5 Soren
Blirup-Jensen,6 Anders Carlstrom,7 Francesco Dati,4 Anthony Milford Ward,8 and Per Just Svendsen6
Quality-control surveys inrecent years, in venous parts of the
world, have shown poor between-laboratoryagreement for
measurements of plasma proteins. Despite the existence of
international reference materials distributed by the World
Health Organization, standards produced by diagnostics
manufacturers and professionalorganizationsdiffer signifi-
cantlyintheirascnbedvalues. The reasons for thisare com-
plex but indude poor availability of the primary matenals,
confusionabouttheir use, and the fact that their turbidity on
reconstitution precludes their use in modem optical immu-
noassays. This unfortunate situation led to an important ini-
tiative to producesufficient quantities of a widely available,
optically dear secondary reference material for plasma pro-
teins that could be used worldwide by manufacturers, profes-
sional organizations,and laboratories. Here we present an
overview on howthe laboratorycommunity,includingmanu-
facturers,clinicallaboratories, professional societies, and reg-
ulators, has reached what we consideris a successfulcon-
clusionto a difficultproblem.
Indexing Terms: standardization/caiibration
Unlike assays of ions and some small molecules for
which the chemical and physical characteristics are well
understood, measurement of large and complex proteins
and peptides depends entirely on the comparison of a
test sample with a reference material. Several different
primary reference materials for serum proteins have
been produced in the last 20 years and issued by the
World Health Organization (WHO).9 A large number of
1hnstitute for Cancer Studies, St. James’s University Hospital,
Beckett St., Leeds LS7 9TF, UK (address for correspondence). Fax
mt +44-532-429886.
for Blood Research, Scarborough, ME.
3Greensboro, NC.
4Behringwerke AG, Marburg, Germany.
5Hospicea Civils de Lyon, Lyon, France.
6Dako A/S, Glostrup, Denmark.
7Danderyds Hospital, Danderyd, Sweden.
8Protein Reference Unit, Sheffield, UK
Produced by the International Federation of Clinical Chemistry
Committee on Plasma Protein Standardization.
9Nonstandard abbreviations: RPPHS, Reference Preparation
for Proteins in Human Serum (CRM 470); WHO, World Health
Organization; IFCC, International Federation of Clinical Chemis-
try; BCR, Community Bureau of Reference of the Commission of
the European Communities; CRM, Certified Reference Material;
CAP, College of American Pathologists; CDCP, Centers for Dis-
ease Control and Prevention; USNRP, US National Reference
Preparation (for human serum proteins); WHO 6HSP, WHO Ref-
erence Preparation for Six Human Serum Proteins; and RPSP,
Reference Preparation for Serum Proteins (CAP).
Received October 18,1993; accepted March 8, 1994.
secondary reference materials produced by professional
and commercial organizations are in use worldwide, to
which values have been assigned by various methods in
relation to the primary materials. Unfortunately, for
reasons that are unclear, the values for some proteins
vary by as much as 100% among different secondary
reference materials. These variations in analyte values
have been evident in quality-assurance surveys, both in
the US and in Western Europe (1, 2). This poor state of
affairs is in part attributable to a combination of confu-
sion about the use of the primary materials; widespread
use of secondary materials, the values of which have
drifted from those of their primary counterparts; poor
availability of the primary materials; and the unsuit-
ability of older preparations for use with more modern
optical measurement systems. Obviously, the use of a
single, internationally agreed upon reference material
should substantially reduce the between-laboratory
variability, especially if a precise method of value trans-
fer from primary materials to operating reference ma-
terials is used.
For these reasons, the International Federation of
Clinical Chemistry (IFCC) Committee for Plasma Pro-
tein Standardization in 1989 began the process of pre-
paring, characterizing, and calibrating a new interna-
tional secondary matrix reference preparation for 14
plasma proteins: transthyretin (prealbumin), albumin,
a1-acid glycoprotein (orosomucoid), a1-antitrypsin (a1-
protease inhibitor), ceruloplasmin, haptoglobin, cr2-mac-
roglobulin, transferrin, C3, C4, IgG, IgA, 1gM, and C-re-
active protein. The material was certified by the
European Community Bureau of Reference (BCR) as a
Certified Reference Material (CRM 470) in mid-1993
and is to be co-released by the College of American
Pathologists (CAP) in mid-1994. CRM 470 is intended
for use as a secondary matrix reference material, from
which values will be transferred to working calibrants
and controls for immunoassay of serum proteins (3).
PrevIous Reference MaterIals
In 1967 Rowe et al. (4, 5) prepared a pool of serum to
serve as the first International Standard for plasma
proteins-the immunoglobulins (4). Part of this pool,
batch 67/86, became the WHO Immunoglobulin Stan-
dard; filled into ainpoules, it was lyophilized at the Na-
tional Institute for Biological Standards and Control in
London. The remainder, processed by the Weilcome Re-
search Laboratories, Beckenham, UK, using a slightly
different procedure, subsequently became batches 67/95,
CUNICAL CHEMISTRY, Vol. 40, No. 6, 1994 935
67/97, and 67/99. Batch 67/86 was defined as having 100
units per ampoule for each of the three iinmunoglob-
ulins and should be thought of as the primary reference
material for immunoglobulins with values in Interna-
tional Units (IU). Values for the proteins in 67/95 and
67/97 were derived by direct comparison with the first
batch (67/86), by means of radial immunodiffusion. Val-
ues for 67/99 were calculated from the mean weight of
the ampoule contents of 67/99 and 67/86. These three
lots are thus secondary reference materials. Mass val-
ues were subsequently ascribed to the primary reference
preparation for IgA, IgG, and 1gM (67/86) by 10 expert
laboratories using immunochemical measurements
against purified proteins. Despite the wide variance
among laboratories, mean values were allocated none-
theless (6).
In 1973 the WHO and the International Union of
Immunological Societies proposed the production of a
new, transparent, stable, lyophilized, pooled human se-
rum to act as a calibrant for several clinically useful
serum proteins. Five candidate preparations were pre-
pared, one of which was chosen as a reference material
for human serum proteins. One-half of this material was
lyophilized in vaccine vials (1.5 mllvial), sealed with
rubber stoppers, and stored at the Centers for Disease
Control and Prevention (CDCP; Atlanta, GA) as the US
National Reference Preparation (USNRP). The remain-
ing half was lyophilized in glass vials (1.3 mLdvial) and
sealed by fusing the glass. The latter became the WHO
Reference Preparation for six Human Serum Proteins
(WHO 6HSP) (6). International Units were arbitrarily
assigned to WHO 6HSP for six proteins (albumin, a1-
antitrypsin, ceruloplasmin, a2-macroglobulin, transfer-
rin, and C3c-making it, therefore, the primary refer-
ence material for these proteins in IU) and, by transfer
with use of single radial immunodiffusion, from WHO
67/86 for the immunoglobulins (for which it is a second-
ary reference material) (7).
Mass concentration values were subsequently as-
signed to the USNRP by 24 expert laboratories (7, 8).
For the immunoglobulins this resulted in a unit-to-mass
conversion factor that differed somewhat from that de-
scribed by Rowe et al. in 1972 (5) for WHO 67/86, al-
though the difference is surprisingly small considering
the improvements in technology in the intervening pa-
riod. The USNRP is thus de facto the primary reference
material for values ascribed in mass units for all the
proteins contained therein (the earlier allocation of
mass values for immunoglobulins to 67/86 can be con-
sidered as superseded by the USNRP as a result of
improved techniques).
Anew reference preparation produced by CAP, the
Reference Preparation for Serum Proteins Lot 1(RPSP-
1), was calibrated at the same time as the USNRP.
RPSP-1, arecalcifled and delipidated plasma, was used
as a reference material by many manufacturers and
laboratories until the supply was depleted. CAP subse-
quently prepared RPSP-2 and RPSP-3; in each case,
values were assigned by consensus in comparison with
the preceding reference material (9). Over the years, the
values drifted somewhat from those originally assigned
from the USNRP.
In 1990 the CAP Standards Committee recognized
that stocks of RPSP-3 would be depleted months or
years before the RPPHS became available. At the sug-
gestion of manufacturers of immunochemical reagents
and instruments, to minimize further changes in value
assignments, CAP decided to use the same primary ref-
erence materials and the method for value transfer
planned for the RPPHS. Mass concentrations were so
assigned; however, the CDCP conversion factors for In-
ternational Units were used rather than International
Units derived from the WHO standards (as in the case of
the RPPHS). RPSP-4 has been available from CAP since
early 1991.
New International Reference Preparation for Proteins In
Human Serum (RPPHS)
Since the release of the WHO International Reference
Standard for Immunoglobulins (67/86), WHO 6HSP,
and USNRP (lot no. 12-0575C) by the CDCP, much has
been learned about the requirements for reference ma-
terials to be used in modern optical immunoassay sys-
tems. After careful evaluation of existing reference ma-
terials,investigators feltthat certain important criteria
must be fulfilled in the preparation of the new RPPHS.
Of major importance is the requirement that the
RPPHS behave in assay systems similar to the serum
samples presented for routine testing. The proteins pre-
sent within the material should thus, if possible, be in a
similar physical state to those in fresh serum and show
no alteration on storage. This was achieved by using
naturally clotted serum (avoiding the use of thrombin,
which may cause protein degradation, and clotting of
anticoagulated plasma, which produces inconsistent
turbidity with time) and by adding protease inhibitors
before lyophilization to ensure preservation during pro-
cessing. For the most part, the early reference materials
for plasma proteins were more turbid than fresh serum,
and this turbidity tended to increase with time over
prolonged storage because of precipitation of residual
fibrinogen incompletely removed by recalcification of
plasma. The effect was a decrease in the signal-to-noise
ratios and an accompanying decrease in precision in
nephelometric and turbidimetric assays. Optimal clar-
ity in the RPPHS was achieved by collecting blood from
volunteers after an overnight fast; allowing spontane-
ous clotting of the blood in glass; rejecting any donations
that were visibly turbid, jaundiced, or hemolyzed; and
absorbing the remaining lipoproteins with micropartic-
ulate silica. This procedure was extremely effective;
however, it caused a significant decrease in overall vol-
ume and a reduction in concentration of those analytes
with an affinity for the silica particles, such as C4 and
1gM.
Allotypic differences in serum proteins resulting from
racial and regional variations have become an impor-
tant issue in the management of analytical data for
serum proteins in recent years. Thus, demographic in-
formation about the donors to the RPPHS was recorded,
936 CLINICALCHEMISTRY,Vol. 40, No. 6, 1994
including a1-antitrypsin and haptoglobin phenotypes, to
allow a similar donor pool to be reassembled in the
future for the production of further material. Blood
units containing 1gM rheumatoid factor and monoclonal
components that could interfere with immunochemica
assays were excluded. Because of social concerns about
the potential hazards presented by serum pooled from
large numbers of donors, the individual donations were
tested for various infections agents, including those
mandated by law, and excluded if found to be reactive.
As a result, considerably more attention has been de-
voted to the constitution of the new reference prepara-
tion than to any previous material.
Ideally, primary reference materials for which all val-
ues are assigned against purified and highly character-
ized proteins are desirable. However, given the avail-
ability of only a few such proteins and the urgent need
for a new international reference material, the Commit-
tee decided in 1989 to proceed with development of a
preparation calibrated against the relevant WHO ma-
terials for International Units (IU) and against the best
available materials for mass/volume units. Methods for
purifying transthyretin, a1-athd glycoprotein, and trans-
ferrin had previously been developed by a Working
Group on Plasma Protein Standardization of the IFCC.
Proteins prepared by the University of Copenhagen,
using these protocols, were used for value transfer to
RPPHS.
As has been known for years, the original material
used for assigning mass values to USNRP for a1-anti-
trypsin has been superseded by modern preparations
that give very different calibration values. Indeed, the
use of these latter preparations has been the major
cause of the marked between-calibrant variation for this
protein. It was thus decided to use a new preparation of
a1-antitrypsin prepared by the Clinical Chemistry Lab-
oratory, Malmo General Hospital, Malmo, Sweden. For
C-reactive protein, the WHO reference material was
used, i.e., 1 IU 1 mg. USNRP (lot no. 12-0575C), from
the CDCP, was used for the assignment of mass/volume
units for the remaining proteins.
Preparationof RPPHS
Extensive testing of the procedures to be used for the
final lot of the RPPHS was undertaken with several
pilot batches. These procedures were shown to result in
a very clear material that could be used in all common
methods of immunoassay. Aclerated degradation
studies showed no significant change in protein concen-
tration in samples of the lyophilized material stored at
45C#{176}for 1 year. All procedures and data for the final lot
were documented in detail to permit the reproduction of
similar material when a new lot is needed (10). The
steps in preparation were as follows:
1) Fresh serum, derived from naturally clotted whole
blood (-175 mL per donor), was collected from several
hundred healthy individuals in five European cities. So
that asimilar donor pool could be assembled in the
future, demographic data for each donor were recorded,
including country of origin, race, age, gender, weight,
and blood group.
2) The individual collections were tested for H1V-1
and -2, HTLV-1, hepatitis B surface antigen, and hepa-
titis C antibody, with test materials approved by the US
Food and Drug Administration, Washington, DC. They
were also tested for the presence of rheumatoid factor,
monoclonal immunoglobulins, and other abnormalities
identifiable by serum electrophoresis. Phenotyping was
performed for a1-antitrypsin and haptoglobin. The indi-
vidual collections were examined for hemolysis, hyper-
bilirubinemia, and turbidity. All collections that
showed abnormalities or possible interfering substances
were excluded.
3) The collections were preserved by the addition of
sodium azide, then frozen and shipped to the central
processing center. The individual collections were
thawed, pooled, and delipidated with fumed micropar-
ticulate silicon dioxide, and then stabilized with addi-
tiotial sodium azide, aprotinin, and benzamidine. Pure
C-reactive protein was added to a final concentration of
-40 mg(L. The material was buffered to pH 7.2, and
subjected to sterile ifitration. Vials were filled with 1.00
mL of the material, freeze-dried, and sealed.
Value Assignments
Analyses for value assignment were performed by 27
laboratories in Europe, the US, and Japan, according to
arigorous protocol designed to test the appropriateness
of the candidate preparation as well as the performance
of the collaborating laboratories (Table 1). Multiple di-
lutions of the RPPHS and of the relevant primary ref-
erence materials were made, such that all assays were
within the same assay range for both materials. Linear-
ity over the range of assays and regression through zero
were required (11) to ensure similar behavior of the
materials (i.e., absence of matrix effect differences) and
lack of antigen excess. The slopes of the regression lines
were used to assign the values. A trial exercise in value
assignment clearly showed greatly improved precision if
all reconstitutions and dilutions were weight-corrected
with a sensitive balance. Therefore, all reconstitutions
and manual dilutions used in the value assignment
were done in this way.
The values assigned to the RPPHS in some cases
significantly differ from those in previous reference ma-
terials, except for RPSP-4 (currently available from
CAP).’#{176}The most significant changes from the mass
values assigned to RPSP-2 and RPSP-3 involve trans-
thyretin, a1-acid glycoprotein, a1-antitrypsin, and 1gM.
These changes range from 10% to 40%. In addition, the
relationships of mass values to IU are different in the
RPPHS, because the IU values were assigned directly
against WHO materials rather than against USNRP.
The evidence from the value assignment exercise sug-
‘#{176}However,referenced against RPPHS, the values assigned to
RPSP-4 for a1-antitrypsin and tranathyretin should be -10%
higher (assigned values, 1.60 and 0.26 g/L, respectively; referenced
against RPPHS, 1.75 and 0.28 g/L, respectively).
USNRP,
lot no.
12-0575C Pure
proteIns
mg/L
C-reactive
protein, Complement, WHO
WHO WHO lot no. lmmunoglobullns 6HSP,lot
85/506 5/4 WHO 67/86 no. 4/2
.
.
ProteIn
Transthyretln
Albumin
a1-Acidglycoprotein
a1-AfltitrypSifl
Ceruloplasmin
a2-Macroglobulln
Haptoglobin
Transfemn
C3/C3c
C4/C4c
C-reactive protein
IgG
IgA
1gM
.
.
C
C
S
IU/L
.
.
S
S
Table 1. InternatIonal reference preparations used In value assignment of the RPPHS.
CLINICALCHEMISTRY, Vol. 40, No. 6, 1994 937
gests that the IU values in USNRP (and RPSP-4) are
incorrect and should not be used. Although WHO 6HSP
and USNRP are part of the same pool, WHO 6HSP was
filled with 1.3 mL (to be reconstituted with 1 mL),
whereas USNRP was filled with 1.5 mL (to be reconsti-
tuted with 1 mL). There should thus be a 14% difference
in protein concentration between the two materials.
This difference was confirmed in the current value as-
signment exercise, with a mean difference for the three
immunoglobulins of 14.3%. If, on the other hand, the
IU/mL values declared on the package inserts are com-
pared, the average deviation is 8.7%; only in the case of
transferrin and the immunoglobulins is it about 14%.
The concentrations of ceruloplasmin, C4, and 1gM are
low in the RPPHS, relative to those in the fresh normal
serum, a result of their strong affinity for the silicon
dioxide used in delipidation. This is particularly impor-
tant for C4 and ceruloplasmin, the concentrations of
which are at the lower limits for assay in some instru-
ments (e.g., the Beckman rate nephelometers).
Availabilityand Use
RPPHS has been released in Europe by the Commu-
nity Bureau of Reference of the European Economic
Community (10). The material has been approved by
the US Food and Drug Administration for distribution
in the US by CAP.
The IFCC Committee intends that the RPPHS be
used as a serum-based reference material for transfer of
values to tertiary materials (calibrants and controls)
and not for direct use in laboratory assays. The current
lot should last for several years if used in this way. The
Committee strongly recommends the use of a value-
transfer protocol similar to that used for assignment of
values to the RPPHS (10). The important aspects of this
protocol include weighing all reconstitutions and dilu-
tions; assaying several dilutions of each material, with
dilutions made so as to be in the same assay range for
the different materials; assaying replications of sam-
ples; and, more important, performing runs on multiple
days, each with calibration of the instrument. Because
of the experimental design and the simplified statistical
analysis, linear regression through the origin was used.
The protocols and statistical analysis used for the value
assignment of the RPPHS are available upon request
from BCR (Commission of the European Communities,
Directorate General for Science, Research & Develop-
ment, DG X11/C/5, Measuring & Testing Programme,
Rue Montoyer 75, B-1040 Brussels, Belgium) or CAP
(325 Waukegan Rd., Northfleld, IL 60093-2750).
Conversion to the new RPPHS will result in inconve-
nience and possible confusion to users of protein data.
Significant changes in reference values will occur for
some proteins (notably, 1gM, a1-acid-glycoprotein, a1-
antitrypsin, transthyretin, and transferrin) if they were
assigned with some older reference materials. These
changes have already been made in the calibration of
CAP RPSP4 (see footnote 10). Reassignment of refer-
ence ranges will thus be necessary, either through anal-
ysis of new reference groups or by the use of conversion
factors supplied by manufacturers of commercial pro-
tein calibrants. The Committee is embarking on a proj-
ect to establish reference ranges, based on this material,
for populations in Europe and the US. Valuable work
has already been done in this regard by Nordkem (Den-
mark).
We can hope that the use of a common calibrator
worldwide for serum protein analysis will result in a
demonstrable improvement in overall performance
among laboratories and kits. However, the innate me-
lecular heterogeneity of proteins and the changes that
occur in disease will ensure that the problem of accurate
protein measurement will never be completely solved
(1). It is the intention of the Committee to assign values
for additional proteins to the RPPHS as time and funds
allow. The BCR has already funded a project to assign
938 CLINICAL CHEMISTRY, Vol. 40, No. 6, 1994
values for a1-antichymotrypsin and Kand Alight chains
of immunoglobulin. Important proteins for future con-
sideration should include the immunoglobulin sub-
classes.
We acknowledge the financial support of the BCR and the con-
tribution made by many diagnostic companies, notably Behrung
Diagnostics, Beckman Instruments, and Dako Corp. We thank the
IFCC Scientific Division for their encouragement and support and
CAP for their cooperation. We thank the following for their help in
this project: Stephen Goodall, The General Infirmary, Leeds, UK;
Thomas Ledue, Foundation for Blood Research, Scarborough, ME;
Elizabeth Colinet and Christos Proflhis, Brussels, Belgium; Path-
cia Gembala, CAP; and Robert Nakamura, Scripps Clinic and
Research Foundation, La Jolla, CA.
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