Standardization
Hormonal Assay
STANDARDIZATION
The term “standardization is used to describe
the process by which comparable results are
obtained with each laboratory method of
calibration, traceable to a reference
measurement procedure.
This requires
A clearly defined analyte
A reference measurement with clear definition in
International system of units (System International
(SI)).
HARMONIZATION
In case if the such a reference material that
clearly can be defined with SI, laboratories can
obtained agreement and comparability among
measurement procedure with some agreed
reference material like purified biomarkers,
single-donor blood material, pooled patient
sample or a manufacturer’s calibrator.
This can be achieved through a single method or
with different method with an all methods
defined mean.
STANDARDIZATION
The aim of standardization is to obtain identical
and correct results with different methods. The
expression “harmonization” is often used as an
alternative to standardization.
However, while different harmonized methods
may provide similar results, they may all be
biased.
STANDARDIZATION
Standardization is feasible if a standard and a
reference method are available.
The reference method should preferably be a
definitive method.
In practice mass spectrometry (MS), which
presently can be used for relatively small
molecules like steroid and thyroid hormones and
also for some peptide hormones is the reference
method.
STANDARDIZATION
Most hormone determinations are performed
by immunoassay but MS is increasingly used for
routine determination of steroid hormones.
Standardization of immunoassays is challenging
and results from quality assessment show that
for many methods, between-method variation is
not yet on an adequate level.
Problems for standardization
Hormone concentrations
Standardization of hormone determinations is
complicated by a number of problems.
First, the concentrations of hormones in
circulation are very low in comparison to those
of other similar substances.
Hormone concentrations
In healthy subjects, the concentrations of most
peptide and protein hormones are in the range
1–50 pmol/l.
The concentrations of the most abundant serum
protein, albumin, is about 0.7 mol/l, which is
10–100 million fold that of typical protein
hormones.
Hormone concentrations
The concentrations of steroid hormones are in
the range 20–300 pmol/ l for estradiol and to
50–500 nmol/l for cortisol.
These should be compared to the concentration
of cholesterol, 5 mmol/l, which is 10,000–
100,000,000–fold higher than those of steroid
hormones.
Hormone concentrations
It is inevitable that the presence of a huge
excess of similar substances affects hormone
determinations by causing nonspecific
interference.
Considering these facts, it is amazing how
specific and sensitive immunoassays are.
Hormone heterogeneity
Most peptide and protein hormones occur in
different forms in circulation, e.g., 22 and 20 kD
growth hormone, intact hCG and its free b
subunit (hCGb) or intact paratahormone and
hormonally inactive, truncated forms.
In order to standardize assays for such
hormones, it is necessary to define which forms
should be measured and to use antibodies that
detect relevant epitopes.
Hormone heterogeneity
Genetic polymorphism of protein hormones is
fairly common, this can affect the assay in
various ways.
A variant of luteinizing hormone (LH), which
differs with respect to two amino acids (Trp8Arg
and Ile15Thr) is not detected by some
antibodies.
Cross-reacting hormones and binding proteins
Standardization of steroid hormone
determinations is complicated by the occurrence
of closely related forms that cross react with the
antisera used. Some antibodies to hCG also
recognize LH and vice versa.
Specific assays can be developed by careful
selection of epitope reactivity of the antibodies
used.
Cross-reacting hormones and binding proteins
The difference in structure between many
steroid hormones, e.g., estradiol and estrone or
testosterone are actually very small and
therefore, some cross-reactivity between
related hormones is inevitable.
Cross-reacting hormones and binding proteins
Most steroid hormones occur in circulation in
complex with steroid-binding proteins that
compete with antibodies.
Many peptide and some protein hormones also
occur in complex with specific binding proteins
in circulation.
Cross-reacting hormones and binding proteins
With the recent advances in MS technology,
these methods are now increasingly used for
routine determinations.
While the cost of MS still is higher than that of
immunoassays, the better validity of MS often
justifies its use.
A misleading result of an immunoassay often
causes high additional costs by leading to
unnecessary imaging and other examinations.
Matrix effects
The composition of the sample affects the
reaction between antigen and antibody and thus
the results of hormone determinations.
Therefore, the calibrators should be prepared in
the same matrix as the sample, usually serum or
plasma.
Matrix effects
Thus it is obvious that preparation of the matrix
for the calibrators is a crucial step in the
development of an immunoassay.
Ideally, the calibrators are prepared by spiking
serum devoid of the analyte with standards of
desired concentrations.
Manufacturers seldom provide information
about how this has been done.
Matrix effects
A common matrix problem is caused by human
antibodies against animal immunoglobulins that
are present at such concentrations that they
disturb the assay.
Circulating antibodies against ovine and bovine
immunoglobulin are actually quite common, as a
result of the ingestion of animal milk and meat.
In most cases the interference can be prevented
by use of animal immunoglobulin in the assay
buffer.
Matrix effects
While the composition of serum (and plasma) is
fairly constant, that of urine varies considerably.
Due to variation of urinary flow rate, the density
of urine is highly variable.
The content of proteins, steroids and other
solutes varies accordingly.
Therefore, the measured concentrations of
analytes in urine is also corrected accordingly.
Matrix effects
The estimation of hormones in urine is also
complicated by the variation in pH, salt and
various metabolites concentration and presence
of steroid conjugates.
Therefore, only few immunoassays have been
validated for analysis of urine samples.
