Implementing Reticulocyte Hemoglobin Into Current Hematology Algorithms

A Systemic Literature Review

Cory R. Lundgren, MLS(ASCP)


Am J Clin Pathol. 2022;158(5):574-582. 

In This Article

Abstract and Introduction


Objectives: This systematic review investigates reticulocyte hemoglobin's capabilities in screening iron deficiency and iron-deficiency anemia with and without comorbidities.

Methods: Participant background and laboratory characteristics were extracted from 14 unique studies between 2015 and 2022. Hemoglobin, reticulocyte hemoglobin, and mean cell volume (MCV) values were used in a meta-analysis for iron-deficiency anemia with no secondary conditions. Mean laboratory values for each patient population were calculated and then used to determine sensitivity, specificity, and the area under the curve (AUC) for iron deficiency and iron-deficiency anemia. The ferritin and reticulocyte hemoglobin ranges were calculated using the mean values.

Results: The meta-analysis demonstrates that hemoglobin and MCV values do not significantly differ between studies, unlike reticulocyte hemoglobin values. The reticulocyte hemoglobin range is smaller than ferritin for the controls, iron deficiency, and iron-deficiency anemia. Reticulocyte hemoglobin values less than 26 pg can distinguish iron-deficiency anemia, while 26 to 31.5 pg can distinguish iron deficiency, with an AUC of 0.889. The sensitivity and specificity are 92.3% and 100% for iron-deficiency anemia, 100% and 81.5% for iron deficiency, and 94.4% and 71.4% for both, respectively (reference range, <31.5 pg).

Conclusions: Reticulocyte hemoglobin is potentially a quick screening test for iron deficiency and iron-deficiency anemia.


Iron deficiency is a frequent issue worldwide and has the potential to cause iron-deficiency anemia when chronic. This process is generally slow and occurs through three steps, where specific laboratory tests can help diagnose the condition.[1] Unfortunately, current diagnostic algorithms using a CBC and an iron panel only detect iron deficiency in the final stage, where there is a significant depletion in body storage of iron. An iron panel can be used to screen healthy individuals before the onset of anemia but is considered inappropriate and, even when ordered correctly, may require 24 to 48 hours for results. A possible solution to this problem is measuring reticulocyte hemoglobin (retic-Hb), which may act as a rapid screening test for absolute iron deficiency. This possibility is due to reticulocytes constituting 1% of all erythrocytes and representing the current erythropoietic status of the bone marrow.[2] This systematic literature review explores several studies to determine if this test is acceptable for screening healthy populations for iron deficiency and iron-deficiency anemia. In addition, this review examines retic-Hb's differential power through studies of patients with these conditions plus comorbidities.

The World Health Organization recognizes iron deficiency as the most common nutritional disorder in the world, where iron deficiency is responsible for at least 30% of all anemias.[3] Iron deficiency is not just a problem in developing countries, given the high prevalence in developed nations. For instance, studies demonstrate that 2% of adult men, 9% to 12% of non-Hispanic White women, and 20% of Black and Mexican American women are iron deficient.[4] More recent studies project these statistics to be higher as US iron intake has fallen from 1999 to 2018, where intake has decreased by 9.5% in females and by 6.6% in males.[5]

According to Keohane et al,[1] iron-deficiency anemia occurs slowly through three stages. Storage iron depletion is the first stage, where body iron stores are progressively lost, but RBC production is still adequate, leading to no laboratory evidence of developing anemia. Furthermore, with normal erythrocytes surviving 120 days, there are no clinical symptoms of the anemia. Transport iron deficiency is the second stage and occurs due to the shortage of the iron storage pool. This stage normally experiences decreasing hemoglobin production but is still within the reference range. Anemic symptoms may start appearing, such as muscle weakness, but are nonspecific. The final stage is functional iron depletion (iron-deficiency anemia), which occurs due to the depletion of the storage, transport, and functional iron. During this phase, RBCs become microcytic and hypochromic, given the inability to produce hemoglobin. Patients in this stage can experience fatigue, weakness, shortness of breath, pica, pagophagia, and spooning of the fingernails.[6] Those in the second and third stages of iron deficiency may appear healthy and experience no symptoms due to their body compensating for the slow progression.[1]

Diagnosing iron-deficiency anemia traditionally begins by examining the patient's CBC for a decrease in hemoglobin, a decrease in mean cell volume (MCV), or an increase in RBC distribution width (RDW). An iron panel is commonly performed after one or more of these CBC values are found to be abnormal. As previously stated, an iron panel can be used to screen healthy individuals but is considered inappropriate. For instance, the CBC results will be within the reference range during storage iron depletion, while ferritin concentrations often fall to less than 20 ng/mL.[7] Similar characteristics occur in transport iron depletion, where CBC results are still within the reference range but are trending toward abnormal. In this stage, an iron panel could show low ferritin, a low serum iron value of less than 50 mg/dL, and low transferrin saturation of less than 16%.[7] It is not until functional iron depletion that hemoglobin, MCV, and RDW are outside the reference range, which typically leads to the detection of the abnormal iron panel results and the anemia diagnosis.[1]

It is vital to consider comorbidities that appear similar to iron-deficiency anemia when examining a patient's CBC. For instance, conditions such as β-thalassemia minor, anemia of chronic inflammation, sideroblastic anemia, and lead poisoning can display a decrease in hemoglobin and MCV.[1] In these cases, examining the patient's CBC alone will not be specific enough to make a diagnosis, where additional testing such as an iron panel, electrophoresis, or bone marrow biopsy is required.

It is also essential to consider the limitations of the iron panel. For instance, serum iron is low in anemia of chronic inflammation; fluctuates during the day, where it is highest in the morning; and depends on intake.[2] Although it is possible to avoid serum iron variations with an overnight fast, it does place an inconvenience on the patient. Serum iron levels also decrease in infections, inflammation, and malignancy, while the value increases in liver disease.[8] In addition, ferritin is an acute-phase reactant that rises during infections, inflammation, and malignancies.[2] Due to these factors, it is possible to miss an iron-deficiency anemia diagnosis if the patient has an underlying condition.

retic-Hb is a test that may be less affected by comorbidities and more specific to iron-deficiency anemia. According to Ucar et al,[2] reticulocytes are the most common immature erythrocyte released from the bone marrow, where they circulate for 1 to 2 days before reaching maturity. Roughly 1% of all erythrocytes in circulation are reticulocytes, making these cells an excellent representation of erythropoiesis in the bone marrow. Often, retic-Hb concentrations decrease during transport iron depletion, stage 2, which may allow for a diagnosis before the onset of symptoms and CBC changes. Also, since retic-Hb concentrations increase well before overall hemoglobin, the test can monitor patient treatment.[2] Since this test uses the same sample type as the CBC and the same instrument, it allows for less patient blood collection, quicker turnaround times, and a faster diagnosis. Furthermore, this test could further reduce unnecessary testing when implemented into anemia algorithms, thus improving patient and hospital costs.