Peripheral Blood Tests in Iron Deficiency Anemia Diagnosis
The Clinical Utility of Peripheral Blood Tests in the Diagnosis of Iron Deficiency Anemia
Alan Mast, MD, PhD Blackwell Science Publishing Co., 2001, 846-867 pages.
Overview:
Accurate and early diagnosis of iron deficiency anemia in adults is essential because it may be
the presenting sign of a gastrointestinal malignancy. Severe iron deficiency is relatively simple
to diagnose and treat. However, in patients with complicated medical problems the diagnosis can
be difficult. There are numerous tests performed on peripheral blood that have traditionally been
used to diagnose iron deficiency. Of these tests, the serum ferritin is by far the most powerful. A
serum ferritin value less than 12µg/L is a highly specific indicator of iron deficiency. However,
because it is an acute phase reactant, the ferritin may be normal or increased in iron deficient
patients with other medical problems. Thus, some patients may require a bone marrow biopsy or
a trial of iron therapy to differentiate iron deficiency from other causes of anemia. Two
peripheral blood tests for iron deficiency, the soluble transferrin receptor and the reticulocyte
hemoglobin content, have recently become available. These tests are sensitive for iron deficiency
and, when used in conjunction with ferritin, provide a significant advance in the ability to
accurately diagnose iron deficiency using non-invasive testing. The soluble transferrin receptor
is a truncated fragment of the membrane-bound protein. Its concentration is proportional to the
cellular expression of the membrane-bound protein and increases with cellular iron needs and
erythrocyte precursor proliferation. An advantage of this test is that it is normal in patients with
anemia of chronic disease and becomes elevated if they develop iron deficiency, a condition for
which ferritin is particularly insensitive. The reticulocyte hemoglobin content is a measure of
recent iron incorporation into hemoglobin and is an early indicator of both iron-deficient
erythropoiesis and the response to iron therapy. It is reported during routine reticulocyte analysis
on the Bayer ADVIA 120 hematology analyzer. Both of these tests are affected by other
erythrocyte disorders, such as ß-thalassemia, that can produce false-positive test results.
Therefore, the correct diagnosis of iron deficiency requires that these tests be interpreted in the
context of the patient[base ']s overall erythrocyte physiology.
Why is iron deficiency an important condition to diagnose? It is the most common cause of
anemia in both adults and children in the United States, and perhaps in most of the world. Also,
it is often the first indicator of occult blood loss, which is associated with gastrointestinal
malignancy.
Further, given the fairly common shortage of stored blood products, particularly red cells, a
better understanding of iron deficiency allows for more rapid diagnosis and treatment of the
condition, enabling more efficient use of blood products.
There are times when a physician may be faced with answering multiple questions concerning a
given patient, including whether or not to perform a full GI workup for occult blood loss,
determining the cause of the patient's bleeding, and assessing whether the patient really is irondeficient.
As for the prevalence of iron deficiency, it should be understood that the average diet provides
sufficient iron for males, but only marginally adequate iron for menstruating women. Iron
requirements are even higher for women who are pregnant or are lactating. In fact, a recent study
at Washington University in St. Louis, Missouri, found that 17% of female participants, all of
whom were medical students, were anemic. So we can see that even in a population where an
increased awareness of potential iron deficiency could be expected, problems of inadequate
intake persist.
Causes of iron deficiency include blood loss from gastrointestinal disorders, menstruation and
blood donation, decreased iron absorption due to conditions such as inadequate diet or GI
surgery, and increased iron requirement due to pregnancy, lactation, or erythropoietin therapy.
Anemia of chronic disease, which is often difficult to differentiate from iron deficiency, is the
second most common cause of anemia in the United States, and is hypothesized to be caused by
an inability to incorporate iron stores in the hemoglobin.
When we set out to evaluate new tests, it is important to first identify the 'gold standard.' In the
context of iron deficiency, one gold standard test by which other tests can be evaluated is the
Prussian Blue stain of bone marrow aspirate. However, while this method will readily detect the
level of iron stores in the bone marrow, it is an invasive and painful procedure, and is relatively
expensive.
A second gold standard is the measurement of response to iron therapy. This can be an adequate
gold standard, but its efficiency can be complicated by the fact that it requires a compliant
patient who will continue to take the iron therapy despite its side effects. This method also
requires a significant amount of time to find out if a patient is iron-deficient or not, because it
takes one to two weeks for hematocrit levels to rise in response to the therapy.
Besides the potential gold standard tests of bone marrow aspirate and response to iron therapy,
there have traditionally been a number of blood tests used to assess iron deficiency. These
peripheral tests include serum iron, TIBC (transferrin), percent saturation of TIBC, serum
ferritin, free erythrocyte protoporphyrin, peripheral smear, hemoglobin, MCV, and RDW.
Additionally, there are two newer blood tests for iron deficiency that will be examined in more
detail: soluble transferrin receptor and reticulocyte hemoglobin content. Numerous studies have
been conducted comparing the more traditional blood tests for iron deficiency to a gold standard
of bone marrow aspirate evaluation, and these studies appear to reach the same conclusion.
For example Lough, et al.1 studied the peripheral blood tests in 447 patients undergoing bone
marrow aspiration. They concluded that, "Serum ferritin should be retained in the laboratory
investigation of iron deficiency," and that, "Serum iron and iron binding capacity should be
removed from the repertoire."
Another study, by Burns, et al.2 evaluated peripheral blood tests in 301 anemic patients, and
concluded that serum ferritin was clearly the only useful test for diagnosing iron deficiency in
hospitalized patients. Importantly, they also pointed out the main limitation for ferritin: that it is
limited by its low sensitivity.
Finally, one other study from Murthy et al.3 studied the peripheral blood tests in 191 patients and
concluded that serum ferritin was the most reliable non-invasive test for iron deficiency.
Several other studies in the literature have come to the same conclusion - that serum ferritin, at
least in most patients, is the single best test for the diagnosis of iron deficiency, and that other
traditionally used peripheral tests add little additional diagnostic utility to serum ferritin alone.
It is important to note, however, as previously mentioned, that there can be a problem with the
sensitivity of serum ferritin. This is due to the fact that serum ferritin is an acute phase reactant,
leading to increased levels in ill persons. This means that when studying iron deficiency in a
given population using serum ferritin levels, iron status can be accurately determined when
levels are above or below given cut-off points, but that there is a substantial portion of the
population that does not meet either cutoff, resulting in an indeterminate diagnoses.
To examine this limitation of serum ferritin a little more closely, think about a possible study in a
given population in which a ferritin of less than 30 µg/L indicates iron deficiency and a ferritin
higher than 100 µg/L indicates that a patient is not iron deficient. That leaves measures from 30100 µg/L as indeterminate. To determine if patients with indeterminate measures are irondeficient, further testing will be necessary.
One option at this point would be to do a bone marrow aspirate. However, given the previously
discussed drawbacks to this method of testing, it would be preferable to have alternatives. The
two newer peripheral tests of iron status - the soluble transferrin receptor and the reticulocyte
hemoglobin content - may provide these alternatives, helping to avoid bone marrow biopsy.
Looking at soluble transferrin receptor (sTfR), it is important to recall that transferrin is the
protein that binds iron, and that iron is transported into a cell through the transferrin receptor.
When cells require iron, they express a number of transferrin receptors on the cell surface, and
the number of expressed transferrin receptors on a cell is a measure of how iron-starved the cell
is. The soluble form of the transferrin receptor results from what can be thought of as a sort of
random proteolysis. At a certain rate of transferrin receptor expression, receptors are cleaved off
the cell surface and become soluble. The level of these circulating transferrin receptors, about
80% of which originate from erythrocyte precursors, is directly proportional to the amount of
transferrin receptor expressed by the cells. This means that when serum transferrin receptor
levels go up, it is usually due to red cells being starved for iron.
Another factor to consider when studying serum transferrin receptors is that any time there is an
increased reticulocyte count, there will be higher levels of transferrin receptors. Accordingly,
conditions associated with erythropoiesis, such as hemolytic anemia or sickle cell anemia, will
result in higher sTfR. Importantly though, sTfR is not elevated in the anemia of chronic disease.
We conducted a study of 62 patients 4 in whom iron deficiency was confirmed either through
bone marrow aspirate or response to iron therapy. This study showed that ferritin testing with a
low cutoff (<12 ng/ml) was very specific, but had very poor sensitivity. When the ferritin cutoff
was raised (<30 ng/ml), sensitivity was raised to 92% without losing any specificity. Soluble
transferrin receptor in this study was also found to be very sensitive and relatively specific, but
had a low positive predictive value. This low predictive value of sTfR, along with the good
performance of the ferritin test in this population, led us to conclude sTfR should be reserved
mainly for patients with anemia of chronic disease, such as those with rheumatoid arthritis. The
results of this study are summarized in Table 1.
Table 1. Mast, et al. (Clin Chem; 1998)
Sensitivity
Specificity
PPV
NPV
Ferritin-12
25
98
75
84
Ferritin-30
92
98
92
98
sTfR
92
84
58
98
To summarize the use of the soluble transferrin receptor, it is a sensitive test for iron deficiency,
but does not differentiate iron deficiency from other causes of increased erythropoiesis, which
may result in a low positive predictive value. It is, however, useful for differentiation of iron
deficiency from anemia of chronic disease.
Regarding reticulocyte hemoglobin content (CHr), we must first understand that reticulocytes are
the stage of the red cells in which they have almost matured - right after they are released from
the bone marrow but before they have completely matured into a red blood cell. As immature
cells, the reticulocytes still contain RNA and can be differentiated from mature red cells. This
means that you can get a sort of 'snap shot' of recent iron available for incorporation into red
blood cells by using reticulocyte hemoglobin content. The reticulocyte lifespan is typically about
four days, but three of these days are in the bone marrow and only one day is in the peripheral
blood. So, by looking at the reticulocyte hemoglobin content, the measure of iron available for
incorporation at a point three days previously is determined.
Reticulocyte hemoglobin content is useful as an early measure of functional iron deficiency
because the only cells being measured are those recently released from the bone marrow. This
means that CHr is going to be very sensitive in detecting recent iron deficiency, and that it can be
used to indicate whether a given patient is responding to iron therapy more quickly than
hematocrit measures.
We recently sought to assess the clinical utility of reticulocyte hemoglobin content for prediction
of bone marrow iron stores in 87 United States military veterans. In this study, participants' CHr,
ferritin, percent transferrin saturation, and mean corpuscular volume (MCV) were prospectively
measured in patients undergoing bone marrow aspiration. Prussian Blue staining of participants'
bone marrow aspirate was used as the gold standard assay of iron status. The peripheral blood
tests were submitted to be performed as routine tests in the laboratory, meaning that these
samples received no special handling. Additionally, 34 non-anemic medical students were
recruited to use as a comparison group.
Results showed clearly that measures of reticulocyte hemoglobin content were lower in the 21 of
87 study group patients found to be iron-deficient based on the absence of stainable iron in their
aspirate.
The iron-deficient study participants had an average CHr of 26.3 pg, while the medical student
control participants averaged 30.8 pg - about the same as the average of the iron-replete
members of the study group. (Table 2).
Table 2. Comparison of the CHr in iron replete and iron deficient patients to non-anemic
medical students.
Average CHr (pg) CHr Range (pg)
Medical
Students 30.8
28.8-32.9
Iron-Replete
Patients 30.7
22.8-43.7
Iron-Deficient Patients
26.3
21.0-36.4
It was determined that 27 pg was the optimal cutoff value to differentiate iron-deficient from
iron-replete patients in terms of reticulocyte hemoglobin content. Fourteen of 21 iron-deficient
patients had a CHr less than 27 pg, resulting in a sensitivity of 66.7% for this test. Of those irondeficient patients whose CHr measures were not below the cutoff, two had received four units of
packed red blood cells within 24 hours prior to the bone marrow exam, and two were undergoing
oral iron therapy. A fifth iron-deficient patient with a CHr level above the cutoff was caused by
an increased MCV(CHr is determined by MCV multiplied by hemoglobin concentration) present
because of a concomitant vitamin B12 or folate deficiency.
In terms of specificity, only six of 66 iron replete patients had a CHr measure of less than 27 pg a specificity of 91%. Of these six patients, two had ß thalassemia and a third had a clinical
history of iron deficiency anemia.
Data from this study showed, based on receiver operator curve analysis, that reticulocyte
hemoglobin content was the best indicator of iron deficiency when compared to ferritin,
transferrin saturation, or MCV.
In conclusion, when looking at peripheral blood tests for iron deficiency, ferritin is a highly
specific test, but has low sensitivity, and the other tests traditionally used for iron deficiency add
little diagnostic power to that of ferritin alone.
The soluble transferrin receptor and reticulocyte hemoglobin content (CHr) are useful newer
tests for detecting iron deficiency that have higher sensitivity and specificity for iron deficiency
than other traditionally used tests. However, abnormal hemoglobin related to thalassemia can
produce false-positive results for iron deficiency in both tests, and therefore results from these
tests should be interpreted in the context of the patient's overall erythrocyte physiology.
It is hoped that a method of combining measures of ferritin, reticulocyte hemoglobin content,
and soluble transferrin receptor can be developed to give an accurate indication of whether or not
a given patient is truly iron-deficient, independent of performing a bone marrow biopsy.
References
1.
2.
3.
4.
Lough M, Egan EL, O'Cearbhaill HG: Diagnosing iron deficiency in a hospital population. Ir J Med
Sci 989;158:108-109.
Burns ER, Nahum S, Lawrence C, Wenz, B: Clinical utility of serum tests for iron deficiency in
hospitalized patients. Am J Clin Path 1990;93:240-245
Murthy GD, Pasquale D, Chu RC, Lansing LS, Tsan MF, Gershman LC, Chikkappa G: Noninvasive
diagnosis of iron deficiency. Fed Pract 1998;15:13-25
Mast AE, Blinder MA, Gronowski A, Chumley C, Scott MG: The clinical utility of the serum
transferrin receptor in the evaluation of iron deficiency anemia. Clin Chem 1998; 44:45-51