To fast or not to fast?

There is no real consensus as to whether TSAT should be measured from fasting or non-fasting (random) blood samples. Proponents of fasting samples point to the vagaries of diurnal and food/fasting-related changes in serum iron concentration (which will affect TSAT, since TIBC = Fe/TIBC), while those who advocate for random samples point to a single study (discussed below) that showed no added value of a fasting blood sample.

Let’s examine the evidence in turn:

1. Serum iron is increased after a meal

  • Iron concentrations are susceptible to preanalytical factors such as:
    • Within-person biological variability
    • Diurnal variation
    • Diet
    • Exercise
  • Clinical laboratories generally recommend blood collection to be performed in the morning when iron levels are thought to be high, sometimes following either 6, 8 or 12 h of fasting prior to sample collection.
  • Population level retrospective study of pre-analytical effects of blood collection time and fasting on 276,307 serum iron test results:
    • 5 patient categories:
      • Adult male (109,087 results, 39.5%, median age 48)
      • Adult female (157,356 results, 56.9%, median age 45)
      • Teenage males (2181 results, 0.8%, median age 16)
      • Teenage females (3130 results, 1.1%, median age 16)
      • Children < 14 years of age (4553 results, 1.6%, median age 10)
    • Iron concentration results for blood collection times from 06:00 to 22:00.
    • The median iron concentrations were:
      • 18 μM (100 ug/dL) for adult and teenage males
      • 16 μM (89 ug/dL) for adult females
      • 15 μM (84 ug/dL) for teenage females and children
    • Mean serum iron concentrations were observed to be highest through most daytime hours from 8:00 to 15:00 with many of the collection time points being within each other’s 95% confidence intervals.
    • For adults and teenage males, the lowest concentrations occurred at fasting times between 4 and 9 h.
    • Past 12 h fasting, the increasing iron means exceeded those reported for patients who had eaten ≤15 min prior to specimen collection (0 h).
    • Mean iron concentrations at each fasting time deviated < 20% from the overall group means.
  • Study of 17 healthy volunteers:
    • 8 women and 9 men
    • The first blood sample was collected between 8:00 and 8:30 a.m. after an overnight fast.
    • Immediately after blood collection, the volunteers consumed a light meal, containing standardized amounts of carbohydrates, protein, and lipids.
    • Subsequent blood samples were collected at 1, 2, and 4 hr after the end of the meal.

2. Fasting iron indices do not add value to random tests in identifying patients with hereditary hemochromatosis

  • Study of 209 C282Y previously undiagnosed homozygotes with transferrin saturation and unsaturated iron-binding capacity testing performed at the initial screening and clinical examination:
    • In the Introduction, the authors state: “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.”
    • Transferrin saturation and unsaturated iron-binding capacity were performed at the initial screening and again when selected participants and controls returned for a clinical examination several months later.
    • 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).
    • Forty-nine percent of transferrin saturation values increased and 55% of unsaturated iron-binding capacity values decreased with the second fasting sample.
    • There was no difference between the sensitivity and specificity of a fasting transferrin saturation and unsaturated iron-binding capacity (>8 h since eating) compared with a non-fasting transferrin saturation and unsaturated iron-binding capacity for the detection of C282Y homozygotes (>45% for women, >50% for men).

In their Discussion, the authors state:

It has been reported that most C282Y homozygotes have persistent elevations in transferrin saturation, and false-positive test results in non-homozygotes would likely return to normal on the second test. This was the rationale for 2 transferrin saturation tests (first random, second test fasting) before proceeding to more diagnostic tests, including DNAbased testing or liver biopsy. However, this study and others failed to confirm the added value of fasting iron tests compared with random iron tests, and the 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 analytic on the basis of our laboratory analysis of blind replicate samples.

3. What do the guidelines say?

Hepatology. 2011;54:328-43; J Hepatol. 2022;77:479-502; Am J Gastroenterol. 2019;114:1202-1218.; Br J Haematol. 2018;181(3):331-340.
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