Clinical evaluation of novel markers in the diagnosis of anemia.

In current Dutch guidelines regarding diagnostic strategies for anemia (Duth
Society for General Practitioners, Dutch Primary Care Collaboration Agreements)
it is advised to make a first classification in anemia diagnosis based on MCV
and ferritin level. Recent studies show that especially the MCV is not
optimally suited to classifiy different causes of anemia (Leers et al. Int.
Jnl. Lab. Hem 2010; 32, 572-581). Also, the interpretation of ferritin levels
can be problematic when an underlying acute phase respons or inflammation is
present, as this can cause an increase in the ferritin level that is not
related to iron-stores. Because of these draw-backs, it can be difficult to
diagnose iron-deficiency anemia from anemie of chronic disease. Recently,
several novel markers have become available that can possibly aid in making
this distinction. However, the additional clinical value of these markers has
not been evaluated in a randomised prospective fashion. The goal of this study
is to determine whether these novel anemia markers indeed have additional
clinical value in the diagnosis of anemia.
Novel anemia markers comprise both cel-related markers as well as serum-based
markers. The cel-related markers are already reported by the current generation
hemocytometry analyzers. At the Catharina Hospital Eindhoven, Sysmex
hemocytometry analyzers are used. Sysmex analyzers automatically report the
reticulocyte hemoglobin equivalent (RET-HE) and the reticulocyte production
index (RPI) when a reticulocyte count is performed. The RET-HE is an estimation
of the hemoglobin concentration in reticulocytes. As reticulocytes are only
present for 1 to 2 days in circulation, the RET-HE gives insight in the
availability of iron for erythropoiesis in the previous 2 days. When a
decreased RET-HE value is found, this means that iron-supply for erythropoieses
was insufficient for adequate hemoglobinization of reticulocytes in the bone
marrow. This is also referred to as functional iron deficiency. An underlying
inflammation or acute phase response can disrupt the availability of iron for
erythropoiesis. The RET-HE can then give insight in whether this decrease in
iron availability has (already) led to functional iron deficiency. The RPI is a
calculated index that indicates whether the reticulocyte repons on anemia is
adequate or not. When a lower reticulocyte respons is found than expected for
the severity of the anemia, this can also indicate an underlying problem of the
bone marrow.
The novel plasma-related anemia markers include the ratio between ferritin and
transferrin (Transferrin/log(ferritin) ratio, Castel et al. CCLM 50:8 2012).
This ratio is suggested to aid in the diagnosis of iron deficiency anemie when
a low-normal ferritin is found. Both transferrin and ferritin are part of our
routine diagnostic process, and the ratio can be automatically calculated in
the laboratory system when both a transferrin and a ferritin level are ordered.
An additional plasma-marker is the soluble transferrin receptor (sTfR). The
sTfR is correlated to the expression of transferrin receptors on celmembranes;
erytropoietic cells of the bone marrow have the highest expression of these
receptors. When iron-requirements increase, this is accompanied by an increase
in the expression of the transferrrin receptor. The sTfR therefore reflects the
functional iron demand of the bone marrow, while ferritin is a marker for the
total iron reserves of the body. In contrast to ferritin, sTfR is not
influenced by an underlying infection/inflammation. The sTfR/log(ferritin)
ration kan also be used to better distinguish between iron-deficiency anemie
and anemia of chronic disease. By plotting this ratio against the RET-HE, the
so-called 'Thomas plot' is obtained (Thomas en Thomas, Clin Chem 2012 48(7);
1066-1076). This plot devides a patient population in three quarters with
different diagnoses (anemia of chronic disease (ACD), latent iron-deficiency,
iron-deficiency or a combination of ACD and iron-deficiency).
Another novel plasma marker in anemia diagnostics is hepcidin. Hepcidin is the
central regulatory protein in iron homeostasis. It can bind to the
iron-exporter ferroportin on duodenal cells and macrofages. By hepcidin
binding, ferroportin is internalized and degraded, resulting in a decrease in
dietary iron uptake and a decrease of iron availability for erythropoiesis.
Inflammation leads to a increase in hepcidin expression, which can result in
functional iron deficiency. On the contrary, absolute iron deficiency, meaning
the depletion of total iron stores of the body, results in a decrease in
hepcidin, so more iron can be obtained from the dietary intake. Because of
these characteristics, hepcidin is also a marker that could aid in the
diagnosis of ACD versus iron-deficiency anemie. In the current situation, no
routine tests are available for measurement of hepcidin in plasma. The
development of novel methods is ongoing and will hopefully lead to a more
affordable method during the course of this study so hepcidin levels can be
measured.

The objective of this study is to evaluate the additional value of novel anemia
markers (RET-HE, RPI, sTfR, sTfR/log(ferritin), transferrin/log(ferritin)) in a
prospective, randomized study design. Our hypothesis is that the novel anemia
markers can aid in an earlier classification of the anemie, so time before
diagnosis and adequate treatment will be significantly shorter.

This study will start in Octobre 2014 and will end approximately in July 2015,
if sufficient patients are included at that time. The doctor in internal
medicine (Dr. W. Peters) who is responsible for referred patients from general
practitioners or other specialists, selects patient older than 18 years who are
referred because of anemia (or exhaustion). All study patient will be seen by
researcher Dr. N. Wlazlo, who will select eligible patients previous to the
first appointment. Dr. N. Wlazlo will make sure all patients eligible for the
study will recieve an information letter regarding the study at least a week
before their appointment. During the first appointment, additional questions
can be discussed, and the patient can then decide to participate in the study
and sign the informed consent form. The researcher will add the relevant
clinical data of the patient to a SPSS-based case record form. Patients who
participate in the study will be randomly devided, with a 50% chance, over a
'classical' and 'modern' branche of the study. In both branches, both the
regular anemie markers as well as the novel anemia markers will be tested.
However, in the 'classical' branche, the results from the novel anemia markers
will not be available for the doctor and the researcher. In the 'modern'
branche, these results are shown in the electronical patientfile together with
a comment from the laboratory specialist (Dr. K. Coene or Dr. V. Scharnhorst).

We consider patient burden to be very low. Most novel anemia markers can be
analyzed from materials that are already available from the 'classical'
diagnostic approach to anemia. During each of the three blood samplings
performed in this study, one extra tube of 3.5 ml is drawn for possible
analysis of EPO, and/or hepcidin in the future.
We consider patient risk in this study to be very low. Patients in the
'classical' branche recieve diagnostic tests and treatment according to the
current practice at the Catharina Ziekenhuis Eindhoven. Patients in the
'modern' branche have a risk of being treated earlier for iron-deficiency
anemia based on the novel anemia markers. The treatment the patient will then
recieve is equal to the standard treatment for iron-deficiency anemia, possible
side-effects of these drugs are known and accepted in daily practice.