Background of the Invention Hereditary 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).

Genetic screening can be used to detect subjects at risk for hereditary hemochromatosis but is extremely costly.

The conventional preliminary 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.

Summary of the Invention In accordance with a preferred embodiment of the invention, a method is provided for conducting an initial screening of a human subject to determine whether the subject is a candidate for further diagnostic testing for hemochromatosis, the method comprising (a) obtaining a sample of a biological fluid from the subject, and (b) determining the unsaturated iron binding capacity (UIBC) value of the sample; wherein a UIBC value below a selected cut-off value indicates that the subject is a candidate for further testing.

Summarv of Drawings Certain embodiments of the invention are described, reference being made to the accompanying drawings, wherein: Figure 1 shows the relationship between unsaturated iron-binding capacity (UIBC screening test) and transferrin saturation (Transferrin Sat t) in 114 serum samples from randomly selected hospital patients (r = -0.78, p <0.05).

Figure 2 shows UIBC in a random sample of blood donors that have been classified as homozygotes, heterozygotes and normals by genetic testing for the hemochromatosis gene (C282Y mutation of the HFE gene).

Data is presented as mean + standard deviation. There was a significant difference between mean UIBC values in all three groups (p values from analysis of variance - ANOVA with Bonferroni correction).

Figure 3 shows the percentage of homozygotes for hemochromatosis in random blood donors with a serum UIBC

greater than and less than 23 Umol/L. (hh - homozygote, Hh - heterozygote, N - normal). Prescreening with UIBC greatly reduces the number of patients that require genetic testing.

Detailed Description of the 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 coloured complex which can be determined spectrophotometrically.

Ferrozine is a preferred chelator.

In order to make population screening for hemochromatosis feasible, there is a need for an inexpensive test useful as a preliminary screen to identify subjects meriting further investigation.

It is also necessary to establish a reproducible cut-off value for the screening test applied to a population, in order to determine whether any particular subject tested within that population is a candidate for further diagnostic testing for hemochromatosis.

In accordance with a further embodiment of the invention, a method is provided for establishing a reproducible cut-off value for serum UIBC in a population, wherein a UIBC value lower than the cut-off

value indicates that the tested subject is a candidate for further investigation.

The method comprises determining UIBC values of serum samples from a population to be examined, by the method described herein, and analysing these values in relation to the hemochromatosis genotype of the subjects tested. Parameters including TSW and ferritin levels of the samples may be included in further analyses.

The cut-off UIBC value for screening for hemochromatosis is in the range of about 18 to about 31 ymol/L.

It can be seen from Example 2 that in the population studied therein, if the value of transferrin saturation indicative of hemochromatosis is taken, for example, as 55%, then UIBC values lower than 23 ymol/L merit further investigation by more sophisticated diagnostic methods, for example genotyping.

Screening with UIBC to preselect subjects for further testing is a very cost efficient strategy with predicted savings of over 90% compared with using genotyping as the initial test.

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 serum samples from 114 hospital patients.

Total iron was measured using the Unimate 5 reagent system for total iron (Hoffman-LaRoche, Mississauga, Ontario).

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UIBC was determined by a colorimetric micro assay employing ferrozine as chromogen.

The method was a modification of the Unimate 7 system for UIBC (Hoffman, La Roche, Mississauga, Ontario), as follows.

Materials Reagent R1: Buffer containing 250 mM Tris, pH 8.4, 50 mM NaHCO3 and 41 yM 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.

40 yl hemolysis-free test serum was mixed with 200 yl R1 at room temperature in a micro well of an Immulor I microtitre strip on a microtitre plate mixer for 10 minutes at setting 5.

The absorbance (Al) of the solution was read at 550 nm using a Titertek Plus microplate reader.

40 yl R2 was then added to test mixture and mixing was continued for 20 minutes at setting 5 on microtitre plate reader. Absorbance at 550 nm was read again (A2).

Samples of the Unimate 7 calibration standard were similarly treated, and controls without serum were similarly treated to give reagent blank values.

UIBC value (ymol/L) was calculated using the formula (A2 - A1 Reagent blank) - (A2 - A, Sample) UIBC = - x concentration (A2 - A, Reagent blank) - (A2 - A,Calibration of calibration standard) standard TS% was calculated from the formula total iron x 100 TSW = (total iron + UIBC)

UIBC values and TS% values were compared as shown in Figure 1. There was a close negative correlation between the UIBC screening test and TSW (r = -0.78).

TSW values of 45% to 62% have been suggested as the threshold above which hemochromatosis should be suspected. Figure 1 shows that this threshold corresponds to UIBC values lower than 18 to 31 ymol/L.

For example, a TSW higher than 55% (Edwards et al., (1993), N. Eng. J. Med., v. 328, pp. 1616-1620) corresponds to a UIBC value lower than 23 ymol/L.

Example 2 5496 blood samples from voluntary blood donors were assayed for UIBC by the method described in Example 1.

40 samples (0.7%) were found to have UIBC values less than 23 ymol/L.

The donors of these 40 samples were tested for the presence of the C282Y mutation of the hemochromatosis gene by the method of Jouanolle et al. (Nature Genetics, (1996), v. 14, pp. 251-252).

Of the 40 donors, 8 were found to be homozygous for the C282Y mutation, and 15 were heterozygotes. All 8 homozygotes had an elevated transferrin saturation but only 3 had an elevated serum ferritin. The heterozygotes were further tested for the presence of the H63D mutation of the gene by the method of Jouanolle (supra) 8 compound heterozygotes, C282Y/H63D, were identified.

Genotyping of a random sample of 385 donors with a UIBC value >23 ymol/L revealed no homozygotes for the C282Y mutation.

The present invention is not limited to the features of the embodiments described herein, but includes all variations and modifications within the scope of the claims.

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.