Note: Descriptions are shown in the official language in which they were submitted.
219879~
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TEST FOR HEMOC~RQ~ATOSIS
The invention relates to methods for diagnosing
hemochromatosis.
Background of the Invention
He~reditary hemochromatosis is a common autosomal
recessive disease in Caucasians, with a prevalence of
approximately 1 in 300(1 to 4). Excessive intestinal
absorption of dietary iron in homozygotes leads to the
progressive accumulation of iron in tissues. Life-
threatening clinical manifestations, such as liver
failure, diabetes, and heart failure, usually occur after
a latent period of 40-60 years. The diagnosis of
hemochromatosis is often missed, and the disease is
commonly discovered during the management of incidental
illnesses or periodic health examinations(5). Factors
contributing to underdiagnosis include (1) asymptomatic
status of most patients until irreversible tissue injury
has occurred, (2) lack of specificity of symptoms when
present, (3) confusion with aLcoholic liver disease, and
(4) lack of awareness of hemochromatosis, including
appropriate investigations. Early detection and
treatment of the disease can prevent the development of
impotence, heart failure, cirrhosis, and hepatocellular
carcinoma and result in long-term survival similar to the
general population(5-7).
The high prevalence, morbidity and mortality, and
benefit of early diagnosis and treatment make
hemochromatosis a prime target for population
screening(4, 8).
The conventional screening test currently in use to
diagnose hemochromatosis is the transferrin saturation
test, which is a two stage test involving determination
of both serum iron and total serum iron-binding capacity.
2198~9~
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Summary of Invention
The present invention provides, in accordance with
one embodiment, a less expensive and more convenient,
one-step test for screening for hemochromatosis in a
subject by determining the unsaturated iron-binding
capacity (UIBC) of the serum of the subject. The method
is an indirect colorimetric method for measuring the iron
binding capacity in serum.
In accordance with a preferred embodiment, an assay
method is employed wherein a known amount of iron is
added to the serum sample in excess of that required to
saturate any available iron-binding transferrin sites and
the excess unbound iron is measured by interacting it
with a chelator with which it forms a detectable chelate-
iron complex. Preferably, the complex is a colouredcomplex which can be determined spectrophotometrically.
Ferrozine is a preferred chelator.
The screening test may be applied to a general
population or to a specific population with an expected
above-normal prevalence of hemochromatosis, such as
siblings of patients.
EXAMPLES
The examples are described for the purposes of
illustration and are not intended to limit the scope of
the invention.
Example 1
UIBC and percentage transferrin saturation (TS%)
were measured in samples from about 130 subjects
diagnosed with hemochromatosis.
TS% was measured using the Unimate 5 reagent system
for total iron and the Unimate 7 system for UIBC, in
accordance with the manufacturer's instructions (Hoffman-
Ferrozine is a trade-mark
21987~
LaRoche, Mississauga, Ontario). TS% = total iron x 100
(total iron + UIBC).
UIBC was determined by a modification of the method
described in the manufacturer's instructions accompanying
the Unimate 7 reagent system for UIBC (Hoffman-La Roche,
Mississauga, Ontario).
The modified method is scaled down so that reactions
are carried out in microwell plates. This reduces the
amount of sample required and facilitates automatic
reading of the test results by a plate reader.
Materials
Reagent R1: Buffer containing 250 mM Tris, pH 8.4, 50 mM
NaHCO3 and 41 ~M sodium azide.
Reagent R2: Chromogen solution containing 160 mM
hydroxylamine and 20 mM Ferrozine (disodium salt of 3-(2-
pyridyl)-5,6-bis(4-sulphophenyl)-S-triazine).
Hemolysis-free blood samples were collected in iron-
free tubes and serum was obtained from the samples.
Briefly, 40 ~l hemolysis-free serum was mixed with
200 ~l R1 at room temperature in Immulor I microtitre
strips on a microtitre plate mixer for 10 minutes at
setting 5.
The absorbance (A1) of the solution was read at 550
nm using a Titertek Plus microplate reader.
40 ~l R2 was then added and mixed for 20 minutes at
setting 5 on microtitre plate reader. Absorbance at 550
nm was read again (A2).
Samples of the calibration standard provided by the
manufacturer were similarly treated, and controls without
serum were carried out to give reagent blank values.
UIBC value was calculated using the formula
AA (Reagent blank) - ~A (test)
UIBC = x concentration
AA (Reagent blank) - ~A (calibration of calibration
standard) standard
- ~198790
UIBC values and TS% values were compared as shown in
Figure 1. There was a close negative correlation between
the UIBC screening test and TS% (r = -0.78). A TS% value
of greater than 55% indicates a need to carry out further
testing to confirm a diagnosis of hemochromatosis. Using
the UIBC screening test of the present invention, a UIBC
value lower than 23% indicates that the subject requires
further investigation.
References
1. Borwein, S.T. et al., (1983), Clin. Invest. Med., v.
6, pp. 171-179.
2. Edwards, C.Q., et al., (1988), N. Eng. J. Med., v.
318, pp. 1355-1362.
3. Hallberg, L. et al., (1989), J. Int. Med., v. 225,
pp. 249-255.
4. Edwards C.W., (1993), N. Engl. J. Med., v. 328, pp.
1616-1620.
5. Niederau, C. et al., (1985), N. Engl. J. Med., v.
313, pp. 1256-1262.
6. Adams, P.C. et al., (1991), Am. J. Med., v. 90, pp.
445-449.
7. Adams, P.C. et al., (1991), Gastroenterology, v.
101, pp. 368-372.
