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Transferrin saturation is widely considered the preferred screening test for hemochromatosis. Unsaturated iron binding capacity has similar performance at lower cost. However, the within-person biological variability of both these tests may limit their ability at commonly used cut points to detect HFE C282Y homozygous patients.
The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 101,168 primary care participants for iron overload using tansferrin saturation, unsaturated iron binding capacity, ferritin and HFE C282Y and H63D genotyping. Transferrin saturation and unsaturated iron binding capacity were performed at initial screening and again when selected participants and controls returned for a clinical examination several months later. A missed case was defined as a C282Y homozygote who had transferrin saturation below cut point (45 % women, 50 % men) or unsaturated iron binding capacity above cut point (150 μmol/L women, 125 μmol/L men) at either the initial screening or clinical examination, or both, regardless of serum ferritin.
There were 209 C282Y previously undiagnosed homozygotes with transferrin saturation and unsaturated iron binding capacity testing done at initial screening and clinical examination. Sixty-eight C282Y homozygotes (33%) would have been missed at these transferrin saturation cut points (19 men, 49 women, median SF 170 μg/L, first and third quartiles 50 and 474 μg/L), and 58 homozygotes (28 %) would have been missed at the unsaturated iron binding capacity cut points (20 men, 38 women, median SF 168 μg/L, quartiles 38 and 454 μg/L). There was no advantage to using fasting samples.
The within-person biological variability of transferrin saturation and unsaturated iron binding capacity limit their usefulness as an initial screening test for expressing C282Y homozygotes.
The diagnosis of hemochromatosis was previously based on a combined clinical and laboratory assessment that included history and physical examination, elevated transferrin saturation and serum ferritin, liver biopsy, iron removed by phlebotomy, and pedigree studies identifying other family members with iron overload1. Since the discovery of the hemochromatosis gene (HFE) in 19962 most studies from referral centers have shown that > 90 % of typical hemochromatosis patients are homozygous for the C282Y mutation of the HFE gene3. Prior to DNA-based testing, it was assumed that most hemochromatosis patients have elevated transferrin saturation. However, population screening studies have shown that many C282Y homozygotes have a normal transferrin saturation, and may never develop clinical signs and symptoms related to iron overload4–9. Transferrin saturation has been recommended in many studies to be the ideal screening test for hemochromatosis because it is widely available and may be increased even in young adults with a genetic predisposition to hemochromatosis. It has been suggested that transferrin saturation is preferable to DNA-based testing as an initial screening test because it may detect other types of iron overload besides those associated with HFE mutations, and iron deficiency. Screening for iron overload with transferrin saturation may reduce the risks of potential genetic discrimination that some authors suggest is associated with identification of a C282Y homozygote with normal serum iron tests10–12. Important characteristics of a screening test are its reproducibility over time and diagnostic sensitivity.
In this study we sought to determine the variability of transferrin saturation and unsaturated iron binding capacity as well as the impact on their use as a practical and sensitive screening test for hemochromatosis.
The study design and overall results of the Hemochromatosis and Iron Overload Screening Study have been previously reported13–14. Participants were recruited from five Field Centers that serve ethnically and socio-economically diverse populations. The study recruited all participants ≥ 25 years of age who gave informed consent and was approved by all local IRBs. All participants had random testing for serum unsaturated iron binding capacity, serum iron, serum ferritin (without intentional fasting) and were genotyped for the C282Y and H63D mutations of the HFE gene. In this analysis, participants that reported a previous diagnosis of hemochromatosis or iron overload (treated or untreated), were excluded because of the potential effects of phlebotomy or other interventions on the serum transferrin saturation and unsaturated iron binding capacity.
Transferrin saturation was calculated using the serum iron /(serum iron + unsaturated iron binding capacity) and expressed as a percentage. Serum iron and unsaturated iron binding capacity were measured using a ferrozine based colorimetric assay (Hitachi 917 analyzer, Roche Diagnostics/Boehringer Mannheim Corp., Indianapolis, IN). Initial transferrin saturation samples from field centers located in the US were tested at the University of Minnesota Medical Center -Fairview, Minneapolis, MN and those from London, Ontario were tested at MDS Laboratory Services, Toronto, Canada. Initial samples from Toronto, Ontario (n = 2,000) and all follow up Canadian samples were tested in Minneapolis. Method biases were assessed three times yearly using external proficiency testing samples provided by the College of American Pathologists Surveys (Northfield, IL) and using blind replicate samples that were collected from 2% of all participants and analyzed in both laboratories. In addition, comparisons between MDS Laboratory Services and the Central Laboratory were conducted before starting the testing, and 2% of the MDS samples were repeated at the Central Laboratory throughout the study. Internal quality control pools at normal and high iron levels were included with each analytical batch and methods were calibrated using Roche calibrator materials and instructions.
HFE C282Y and H63D were detected in DNA obtained from whole blood ethylenediaminetetraacetic acid (EDTA) samples using a modification of the Invader assay (Third Wave Technologies, Madison, WI) that increases the allele-specific fluorescent signal by including 12 cycles of locus-specific polymerase chain reaction before the cleavase reaction13.
Transferrin saturation and unsaturated iron binding capacity were performed at initial screening and again when selected participants and controls returned for a clinical examination several months later. Clinical examinations were performed on participants with an elevated transferrin saturation and ferritin, all C282Y homozygotes, and control participants (matched for age, gender and race) without HFE mutations and normal transferrin saturation and ferritin (n = 2,285). Initial screening specimens were obtained randomly throughout the day (i.e., without intentional fasting); samples for transferrin saturation and unsaturated iron binding capacity measurements at clinical examination were obtained after fasting (mean time since last meal 13 hours). This study represents modeling based on the HEIRS data; the actual cut points and detection of C282Y homozygotes have been previously reported14.
At cut points for transferrin saturation and unsaturated iron binding capacity (transferrin saturation > 45 % for women, > 50 % for men, unsaturated iron binding capacity is < 150 μmol/L in women, < 125 μmol/L % in men), the potential number of missed C282Y homozygotes was modeled at initial screening and at the clinical examination (Figure 3). A missed case was defined as a C282Y homozygote who had transferrin saturation below cut point or unsaturated iron binding capacity above cut point at either initial screening or at the clinical examination, or both, regardless of serum ferritin. This assumes that for an ideal screening test, a case will have a positive test at the time of each test, and a non-case will have a negative test at the time of each test. Missed cases were expressed as patients missed divided by total patients, rather than total number of tests.
Control participants for the clinical examination had neither C282Y or H63D HFE mutations detected and a transferrin saturation between the 25th and 75th percentile, This selection criteria for the controls results in an apparent “gap” in the transferrin saturation distribution around 40 % (Figure 1).
Fasting and non-fasting samples were compared using Receiver Operating Characteristic (ROC) curve analysis and comparisons of area under the curve.
The HEIRS Study recruited 101,168 participants from February 2001- February 2003. There were 1,261 participants (97 C282Y homozygotes) excluded from this analysis, 1,216 because of a previous diagnosis of hemochromatosis or iron overload and an additional 45 with a missing unsaturated iron binding capacity. There were 236 undiagnosed C282Y homozygotes in this analysis (91 men, 145 women). Non-C282Y homozygotes included 37,004 men and 62,667 women. The median age of all participants in this study was 50 years (range 25 – 100). By self-identified race/ethnicity, the sample included 44 % Caucasian, 27 % African-American, 13 % Asian, 13 % Hispanic, 0.7 % Pacific Islander, 0.7 % Native American and 2 % mixed or unknown race. The C282Y homozygotes were 94 % Caucasian. There were 5 Hispanic and 3 African American C282Y homozygotes. An elevated serum ferritin was found in 86 % of the male C282Y homozygotes (> 300 μg/L) and in 58 % of the female homozygotes (> 200 μg/L).
Analytical variation estimated from a sample of blind replicates at the initial screening visit comprised 1.3% of the total variation in transferrin saturation and 4.4% of the total variation in unsaturated iron binding capacity, and the correlation between replicates was 0.98 for transferrin saturation and 0.99 for unsaturated iron binding capacity. Figures 1 and and22 display substantial variability from the initial screening to clinical exam visit among both C282Y homozygotes and non-C282Y homozygotes. Since the analytic variability is a small percentage, the observed visit-to-visit variability is primarily within-person biological variability. The correlation between initial screening and exam visit values among non-C282Y homozygote men was 0.47 for transferrin saturation and 0.58 for unsaturated iron binding capacity; the corresponding values for C282Y homozygote men were 0.62 and 0.49 respectively. Results for women and estimates from the subset who were fasting at both visits were similar.
There were 209 previously undiagnosed C282Y homozygotes with transferrin saturation and unsaturated iron binding capacity testing done at initial screening and at the clinical examination (Figures 1 and and2).2). The number of missed homozygotes at a clinically relevant cutpoint for transferrin saturation and unsaturated iron binding capacity was assessed (transferrin saturation > 45 % women, transferrin saturation > 50 % men, unsaturated iron binding capacity < 150, < 125 μmol/L men). Sixty-eight C282Y homozygotes (33%) would have been missed at these transferrin saturation cut points (19 men, 49 women, median serum ferritin at initial screening = 170μg/L, first and third quartiles 50 and 474 μg/L), and 58 homozygotes (28 %) would have been missed at the unsaturated iron binding capacity cut points (20 men, 38 women, median serum ferritin at initial screening = 168μg/L, quartiles 38 and 454μg/L). The percentage of missed homozygotes increases as the transferrin saturation cutpoint increases or the unsaturated iron binding capacity cutpoint decreases. Forty-nine percent of transferrin saturation values increased and 55 % of unsaturated iron binding capacity values decreased with the second fasting sample.
A sub-analysis was done which excluded participants initially tested at MDS Laboratories to remove all inter-laboratory variability. All of these participants had their testing done at the Minneapolis site (n = 126 homozygotes). In this sub-analysis, the percentage of missed homozygotes by transferrin saturation (34 %) and unsaturated iron binding capacity testing (29 %) did not differ from the larger sample (n = 199 homozygotes).
The random testing included 29,994 participants who did provide fasting samples. The sensitivity and specificity of a fasting transferrin saturation and unsaturated iron binding capacity (> 8 h since eating) compared to a non-fasting transferrin saturation and unsaturated iron binding capacity for the detection of C282Y homozygotes (>45 % women, > 50 % men) is shown in Table 1. The area under the ROC curves (AUC) were compared for men and women using fasting and non-fasting samples. For men, the AUC for fasting samples was 0.96 (0.92 – 1.0, 95 % confidence interval) and for non-fasting samples was 0.93 (0.89 – 0.97). For women, the AUC for fasting samples was 0.89 (0.83 – 0.95) and for non-fasting samples was 0.91 (0.87 – 0.95). There were no significant differences between fasting and non-fasting samples.
Many advisory documents on screening for hemochromatosis recommend that the initial screening test should be the transferrin saturation6,10,15,16. This is based on the assumption that most iron loaded hemochromatosis patients will have elevated transferrin saturation. Screening with a phenotypic test will detect primarily iron loaded cases requiring treatment regardless of genotype and will also detect iron deficiency. The initial studies on transferrin saturation were performed in tertiary referral centers and subsequent population based studies demonstrated a much lower sensitivity of transferrin saturation for the detection of C282Y homozygotes4,17. Furthermore, elevated transferrin saturation has been often embedded in the case definition which does not allow for an independent evaluation. The unsaturated iron binding capacity has been shown in population studies to be similar in screening performance to the transferrin saturation and can be performed at a lower cost17. However, both of these tests are subject to analytical and biological variability which limit their utility as screening tests for C282Y-linked hemochromatosis. In this study we have demonstrated that at a clinically relevant cutpoint for transferrin saturation and unsaturated iron binding capacity, initial phenotyping with transferrin saturation or unsaturated iron binding capacity could miss approximately 30 % of cases and 40 % of these missed cases had an elevated ferritin. Therefore, it is not simply a matter of non-expressing cases being missed by phenotypic screening.
Within-person biological variability in iron tests has been previously described and diurnal fluctuations have been described primarily for serum iron18–22. Transferrin saturation is a calculated value determined from the serum iron divided by one of the following: total iron binding capacity, unsaturated iron binding capacity plus serum iron, or serum transferrin multiplied by a constant. The higher variability in transferrin saturation compared to unsaturated iron binding capacity may be related to the fact that it is a two step test rather than the single step unsaturated iron binding capacity test. It has been reported that most C282Y homozygotes have persistent elevations in transferrin saturation and false positive tests in non-homozygotes would likely return to normal on the second test23. This was the rationale for two transferrin saturation tests (first random, second test fasting) before proceeding to more diagnostic tests including DNA-based testing or liver biopsy. However, this study and other previous studies have failed to confirm the added value of fasting iron tests compared to random iron tests18,22 and variability in this study was similar between homozygotes and non-homozygotes. Fasting adds a level of complexity and inconvenience to a screening program. As illustrated in this study, the second fasting value is as likely to increase as decrease and regression to the mean is the most likely explanation. Any biochemical test with such wide biological variation is unlikely to be an ideal screening test. In this study, we assume that most of the observed variability was biological rather than analytical based on our laboratory analysis of blind replicate samples.
If the goal of a screening project is to detect C282Y homozygotes (regardless of expressivity), it would seem most advisable to utilize the DNA-based test directly, rather than to perform indirect iron tests to guide the definitive DNA testing. Large population studies in several countries have demonstrated that documented instances of genetic discrimination are essentially nonexistent and the genotypic HFE test is well accepted by participants without long term psychosocial consequences24–26. The cost of the DNA-based test can be relatively modest because it only tests for a single mutation and, given the large sample volumes, can be performed on an automated instrument platform.
Determining the value of screening iron tests for the detection of non-HFE-related iron overload was outside the focus of the present analyses, but other studies have suggested that transferrin saturation is more commonly elevated in C282Y-linked than in non-HFE hemochromatosis1. The latter group of conditions are relatively uncommon and phenotypically and genetically heterogeneous; many of the associated mutations are rare. As a consequence, it is unlikely that genetic tests for non-HFE hemochromatosis-associated genes will become commercially available. Therefore, assessment of non-HFE linked hemochromatosis will likely depend on phenotypic testing and clinical assessment. The design of this study does not allow for an assessment of the operating characteristics of transferrin saturation and unsaturated iron binding capacity for the assessment of iron overload, because this would require a study in which all participants, including those with normal screening transferrin saturation and unsaturated iron binding capacity, would undergo quantitative phlebotomy or liver biopsy. Similarly, estimation of the degree of iron overload by means other than measurement of serum ferritin concentration was beyond the scope of the HEIRS Study.
In summary, both the transferrin saturation and unsaturated iron binding capacity have significant within-person biological variation in C282Y homozygotes discovered through a primary care screening program. This limits their utility as ideal screening tests for HFE-associated hemochromatosis. Other screening approaches such as HFE genotyping followed by measurement of serum ferritin require further evaluation.
The HEIRS Study was initiated and funded by NHLBI, in conjunction with NHGRI: N01-HC-05185 (University of Minnesota), N01-HC-05186 (Howard University), N01-HC-05188 (University of Alabama at Birmingham), N01-HC-05189 (Center for Health Research, Kaiser Permanente), N01-HC-05190 (University of California, Irvine), N01-HC-05191 (London Health Sciences Centre), N01-HC-05192 (Wake Forest University). Additional support was provided by the University of Alabama at Birmingham General Clinical Research Center (GCRC) grant M01-RR00032, Southern Iron Disorders Center (J.C.B.), Howard University GCRC grant M01-RR10284, Howard University Research Scientist Award UH1-HL03679-05 from the National Heart, Lung, and Blood Institute and the Office of Research on Minority Health (V.R.G.); and grant UC Irvine M01 RR000827 from the General Clinical Research Centers Program of the National Center for Research Resources National Institutes of Health (C.E.M.).
Besides any acknowledgments specific to a manuscript, the following should be included: Participating “HEIRS Study” Investigators and Institutions:
Birmingham, AL– University of Alabama at Birmingham:
Dr. Ronald T. Acton (Principal Investigator), Dr. James C. Barton (Co-Principal Investigator), Ms. Deborah Dixon, Dr. Susan Ferguson, Dr. Richard Jones, Dr. Jerry McKnight, Dr. Charles A. Rivers, Dr. Diane Tucker and Ms. Janice C. Ware. Irvine, CA – University of California, Irvine:
Dr. Christine E. McLaren (Principal Investigator), Dr. Gordon D. McLaren (Co-Principal Investigator), Dr. Hoda Anton-Culver, Ms. Jo Ann A. Baca, Dr. Thomas C. Bent, Dr. Lance C. Brunner, Dr. Michael M. Dao, Dr. Korey S. Jorgensen, Dr. Julie Kuniyoshi, Dr. Huan D. Le, Dr. Miles K. Masatsugu, Dr. Frank L. Meyskens, Dr. David Morohashi, Dr. Huan P. Nguyen, Dr. Sophocles N. Panagon, Dr. Chi Phung, Dr. Virgil Raymundo, Dr. Thomas Ton, Professor Ann P. Walker, Dr. Lari B. Wenzel and Dr. Argyrios Ziogas. London, Ontario, Canada – London Health Sciences Center:
Dr. Paul C. Adams (Principal Investigator), Ms. Erin Bloch, Dr. Subrata Chakrabarti, Ms. Arlene Fleischhauer, Ms. Helen Harrison, Ms. Kelly Jia, Ms. Sheila Larson, Dr. Edward Lin, Ms. Melissa Lopez, Ms. Lien Nguyen, Ms. Corry Pepper, Dr. Tara Power, Dr. Mark Speechley, Dr. Donald Sun and Ms. Diane Woelfle.
Portland, OR and Honolulu, HI – Kaiser Permanente Center for Health Research, Northwest and Hawaii, and Oregon Health and Science University:
Dr. Emily L. Harris (Principal Investigator), Dr. Mikel Aickin, Dr. Elaine Baker, Ms. Marjorie Erwin, Ms. Joan Holup, Ms. Carol Lloyd, Dr. Nancy Press, Dr. Richard D. Press, Dr. Jacob Reiss, Dr. Cheryl Ritenbaugh, Ms. Aileen Uchida, Dr. Thomas Vogt and Dr. Dwight Yim.
Washington, D.C. – Howard University:
Dr. Victor R. Gordeuk (Principal Investigator), Dr. Fitzroy W. Dawkins (Co-Principal Investigator), Ms. Margaret Fadojutimi-Akinsiku, Dr. Oswaldo Castro, Dr. Debra White-Coleman, Dr. Melvin Gerald, Ms. Barbara W. Harrison, Dr. Ometha Lewis-Jack, Dr. Robert F. Murray, Dr. Shelley McDonald-Pinkett, Ms. Angela Rock, Dr. Juan Romagoza and Dr. Robert Williams.
Minneapolis, MN – University of Minnesota and University of Minnesota Medical Center, Fairview:
Dr. John H. Eckfeldt (Principal Investigator and Steering Committee Chair), Ms. Susie DelRio-LaFreniere, Ms. Catherine Leiendecker-Foster, Dr. Ronald C. McGlennen, Mr. Greg Rynders, Dr. Michael Y. Tsai and Dr. Xinjing Wang.
Winston-Salem, NC – Wake Forest University:
Dr. David M. Reboussin (Principal Investigator), Dr. Beverly M. Snively (Co-Principal Investigator), Dr. Roger Anderson, Ms. Aarthi Balasubramanyam, Ms. Elease Bostic, Ms. Brenda L. Craven, Ms. Shellie Ellis, Dr. Curt Furberg, Mr. Jason Griffin, Dr. Mark Hall, Mr. Darrin Harris, Ms. Leora Henkin, Dr. Sharon Jackson, Dr. Tamison Jewett, Mr. Mark D. King, Mr. Kurt Lohman, Ms. Laura Lovato, Dr. Joe Michaleckyj, Ms. Shana Palla, Ms. Tina Parks, Ms. Leah Passmore, Dr. Pradyumna D. Phatak, Dr. Stephen Rich, Ms. Andrea Ruggiero, Dr. Mara Vitolins, Mr. Gary Wolgast and Mr. Daniel Zaccaro.
NHLBI PROJECT OFFICE
Bethesda, MD – Ms. Phyliss Sholinsky (Project Officer), Dr. Ebony Bookman, Dr. Henry Chang, Ms. Kristianne Cooper, Dr. Richard Fabsitz, Dr. Cashell Jaquish, Dr. Teri Manolio and Ms. Lisa O’Neill.
NHGRI PROJECT OFFICE
Bethesda, MD – Dr. Elizabeth Thomson.
Dr. Jean MacCluer, Southwest Foundation for Biomedical Research, also contributed to the design of this study.
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