The following is the patient’s complete blood count (CBC) when you see her in clinic:

WBC (109/L)Hb (g/dL)Hct (%)MCV (fL)PLT (109/L)

What’s what: WBC, white blood cell count; Hb, hemoglobin; MCV, mean cell volume; MCHC, mean cellular hemoglobin concentration; RDW-SD, red cell distribution width-standard deviation; platelets, PLT; Normal values: WBC 5-10 x 109/L, RBC 4-6 x 1012/L, Hb 12-16 g/dL, Hct 35-47%, MCV 80-100 fL, MCHC 32-36 g/dL, RDW-SD < 45 fL, platelets (PLT) 150-450 x 109/L

Here are the results of her iron indices:

Is the patient iron deficient?

See next slide for explanation.

Is the patient’s low serum ferritin of 24 ng/ml (mg/L) diagnostic of iron deficiency? Let’s look at the data:

Below are recommended serum ferritin cutoffs to determine iron deficiency:

Cross-sectional studies have examined the sensitivity and specificity of serum ferritin concentrations for diagnosing iron deficiency, using iron staining of bone marrow aspirate as the gold standard:

  • In a study of 203 women, SF concentration cutoff of < 30 mg/L resulted in a sensitivity and specificity of 93% and 75%, respectively
  • In a study of 123 patients, SF concentration
    • < 15 mg/L had a sensitivity of 72% and specificity of 96%
    • < 30 mg/L, had a sensitivity of 92% and specificity of 92%
  • In a study of 20 healthy students SF concentration:
    • < 40 mg/L had a sensitivity of 100% and specificity of 92%.
    • <15 mg/L had a sensitivity of 57% and specificity of 100%
  • In a study of 53 healthy students, SF concentration:
    • < 15 mg/L had a sensitivity of 60% and specificity of 100%
    • < 30 mg/L had a sensitivity of 100% and specificity of 89%

Our patient has a ferritin of 24 mg/L. Based on the data above, she has a pretest probability of iron deficiency of about 90%

Let’s return to the patient’s iron indices:

Is the TSAT normal, decreased or increased?


A normal transferrin saturation (TSAT) is 20% to 45%.

Schematic showing transferrin synthesized in the liver and released into the blood circulation (abstractly represented by the whole field in which transferrin is shown). Iron is absorbed by the gastrointestinal tract and is released into the bloodstream by tissue macrophages (not shown). Iron does not normally circulate freely, but is bound to transferrin. Each transferrin molecule can bind up to 3 molecules of iron. Normally, only about 30% of the iron-binding sites are occupied. Transferrin delivers iron to developing erythroid precursor cells in the bone marrow.

You review the outside labs and note that just 4 months ago, her transferrin saturation (TSAT) was 60-70% on two occasions, including on a fasting sample. Not knowing anything about the patient, what are the three likeliest causes of such a high TSAT?

Schematic from an earlier slide showing transferrin synthesized in the liver and released into the blood circulation. Iron is absorbed by the gastrointestinal tract and is stored in and released by tissue macrophages (upper right). Iron does not normally circulate freely, but is bound to transferrin. Each transferrin molecule can bind up to 3 molecules of iron. Normally, only about 30% of the iron-binding sites are occupied. Transferrin delivers iron to developing erythroid precursor cells in the bone marrow
Schematic showing iron overload state (e.g., hereditary hemochromatosis). Note the increased absorption or iron from the intestines, and the increased release of iron from tissue macrophages into the blood (upper right), owing to an opening of the “floodgates” from reduced hepcidin (gatekeeper) function. As a result, serum iron increases, as does transferrin saturation. Over time, ferritin levels inside cells (especially, macrophages and hepatocytes) increase in order to accommodate storage of the iron. Ferritin is secreted into the circulation at levels proportional to the storage form.

In this case, there is no history of congenital hemolytic anemia (which is associated with ineffective erythropoiesis) or blood transfusion, two causes of iron overload. Therefore you wonder about a diagnosis of hereditary hemochromatosis and you order a genetic screen. The following are the results:

Are these results consistent with a diagnosis of hereditary hemochromatosis?

Either homozygous for C282Y or compound heterozygous for C282Y and H63D qualify for a diagnosis of hereditary hemochromatosis. H63D homozygosity does not.

Is it possible to have iron deficiency in a patient with hereditary hemochromatosis?


The patient has hereditary hemochromatosis, symptoms of iron deficiency and a low serum ferritin. These findings support a diagnosis of iron deficiency in the setting of hereditary hemochromatosis, probably secondary to menstrual blood loss. The patient is sufficiently symptomatic that she wishes to try a course of iron. She is administered IV Feraheme 510 mg x 2. Two months later, her iron indices are repeated:

Should the patient receive a phlebotomy at this point?

Despite the high TSAT, her iron stores are not elevated based on her normal ferritin level. The goal of phlebotomy is to maintain the serum ferritin < 50-100 ng/ml.

Hereditary hemochromatosis is seen more often (and tends to be more severe) in men than women. Some researchers believe that this may be due, in part, to a woman’s monthly blood loss through menstruation. Consequently, iron accumulation is slower in women than men, and the average age of diagnosis for women is approximately 10 years later than in men (usually after menopause).

This case highlights several important points:

  • Even patients with hereditary hemochromatosis may develop iron deficiency from bleeding.
  • The diagnosis of hereditary hemochromatosis may be masked by iron deficiency:
    • In this case, the ferritin was consistently normal.
    • The transferrin saturation (TSAT) may be normal, as seen when the patient was iron deficient.
  • An isolated elevation of TSAT (normal serum ferritin) is a normal finding early on in all patients with hereditary hemochromatosis. This phenomenon is nicely captured in the following letter to British Medical Journal:

BMJ 2011;342:c7251

The following are labs from another patient, in this case a 21 year-old woman, that demonstrate an increased transferrin saturation (TSAT) prior to any increase in serum ferritin:

Hereditary hemochromatosis (HH) genetic screen:

Explaining an early elevation of transferrin saturation (TSAT) in the absence of elevated ferritin:

Whether early or late in the course of hereditary hemochromatosis, the hepcidin-controlled floodgates are wide open, promoting entry of iron into the circulation from the intestine and from storage pools (primarily in macrophages). This leads to excess iron in the blood and an elevated transferrin saturation as we saw in our original patient after her iron deficiency was treated. However, early on there is no demonstrable increase in storage iron (the serum ferritin is normal).
Later in the course of the condition, there is progressive accumulation of storage iron, which is reflected by an increase in tissue iron (stored as ferritin) and a parallel, proportional increase in serum ferritin.

Let’s consider transferrin saturation (TSAT) in more detail:

  • The TSAT is the ratio of the serum iron concentration and the total iron binding capacity (TIBC) expressed as a percentage.
  • Approximately 2% of the adult US population has TSAT > 55%.
  • TSAT reported to have a sensitivity of greater than 90% for hemochromatosis.
  • TSAT is often elevated in young adults with hemochromatosis before the development of iron overload or a rise in ferritin concentration.
  • A common threshold used in screening studies is >45% in women and >50% in men.
  • There is considerable diurnal variations in TSAT (see next slide):
    • Serum transferrin is usually steady with no significant short-term fluctuations.
    • However, serum iron usually fluctuates diurnally and changes acutely depending on dietary iron intake.
    • Some, but not all, authorities recommend a fasting morning sample as a means to overcome this variability.
  • Transferrin is a negative acute protein. Inflammation results in lower TIBC but also lower serum iron, so the net TSAT is often reduced.
  • Transferrin is produced by the liver. Thus, TIBC is often reduced in cirrhosis. The serum iron may be normal or high leading to elevated TSAT.

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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