Reticulocyte Hemoglobin Content: Early Marker of Iron-Deficiency Anemia

Iron deficiency is the most common cause of anemia worldwide as it is the predominant nutritional deficiency.¹ Anemia is defined as hemoglobin of less than 13 g/dL in men and less than 12 g/dL in women, according to the World Health Organization.² Lack of iron leads to suppression of hemoglobin synthesis, which affects the entire body.³ The consequences of iron deficiency anemia include pagophagia, koilonychia, pallor, tachycardia, fatigue, dizziness, shortness of breath, poor concentration, and apathy.⁴

In developing countries, common causes of iron deficiency include malnutrition, poverty, hookworm infections, and schistosomiasis. In the developed world, chronic blood loss most commonly leads to this condition and is frequently caused by heavy menstrual bleeding, colorectal cancer, or gastrointestinal blood loss through inflammatory bowel diseases or peptic ulcers.⁴ Vegan diets and malabsorption conditions also may lead to iron deficiency. Increased metabolic requirements, such as those seen in pregnant women, also may be a cause iron-deficiency anemia.

The gold standard for diagnosing iron deficiency anemia is bone marrow aspiration. However, because this technique is invasive, expensive, and arduous, serologies are typically used to measure iron status. These laboratory markers include ferritin, serum iron, transferrin, and total iron binding capacity (TIBC).⁵ Serum ferritin indicates the storage of iron in the body, whereas serum iron indicates the iron available to be used. Transferrin is a protein that binds iron as it travels throughout the body and TIBC is the quantity of transferrin not bound to iron. Iron deficiency anemia is suspected when serum ferritin and serum iron are decreased but TIBC is increased.² Although these values can be used to diagnose iron deficiency anemia, they are highly influenced by other processes in the body, as shown in Table 1.

Table 1. Influences of Other Factors on Laboratory Markers of Iron Status

Marker	Cause of Increased Levels	Cause of Decreased Level	Meaning of Marker	Disadvantages of Marker
Serum iron	Iron overload	Iron deficiency, inflammation	Amount of iron the body has in total	High variability, neither sensitive nor specific
Ferritin	Acute phase, lymphomas, liver disease	Reduced iron stores	Iron storage	Acute phase reactant
TIBC	Iron deficiency anemia, pregnancy	Hemolytic anemia, hypoproteinemia, liver disease, iron overload	Ability to bind and carry iron	Calculated; only signifies transport needs
Transferrin	Iron deficiency, pregnancy	Inflammation, tumors, hemolysis	Transports iron, dependent on erythropoiesis	Only indicates demand of erythropoiesis, not supply
RET-He	—	Iron deficient erythropoiesis	Functional availability of iron	Not available in every office

Ferritin is an acute phase reactant, meaning it increases during times of inflammation.² The iron becomes trapped inside the cells as a protective mechanism during inflammatory states, leading to increased ferritin values. Because of this protective storage mechanism, transferrin may also be decreased during times of chronic inflammation. Ferritin levels may

not accurately reflect body iron stores in patients with chronic inflammatory conditions or chronic kidney disease.⁵ Serum iron, though helpful to assess current status, fluctuates throughout the day and is highly affected by diet. Physical activity also can cause a temporary inflammatory state leading to altered iron metabolism indicators.⁶ Whether it is chronic inflammation or other acute inflammatory processes, these iron indices are highly variable, suggesting a need for a more direct method of measuring iron availability.

Reticulocyte Hemoglobin Content

Reticulocyte hemoglobin content (CHr) is a diagnostic tool that has shown to be more effective than other measurements of iron metabolism in diagnosing iron-deficiency anemia and is not affected by other factors such as acute or chronic inflammation, infection, malignancy, or liver disease. As immature red blood cells, reticulocytes cycle through the body for 2 days before differentiating into mature erythrocytes. Reticulocytes are the first product of erythropoiesis, thus providing the most accurate and real-time assessment of red blood cell production. In the presence of iron deficiency, the reticulocyte count will decrease as the body does not have adequate components for hemoglobin within the immature erythrocytes. The hemoglobin content of these cells reflects the quality of erythropoiesis rather than just the quantity, and because hemoglobin is composed of iron, it is a direct indicator of iron status throughout the body. Using flow cytometry, hemoglobin content in these cells can be quantified by either reticulocyte hemoglobin equivalent (RET-He) or CHr.³

Reticulocyte hemoglobin content is calculated through the direct cell volume and hemoglobin content of reticulocytes in a H*3 or ADVIA blood analyzer.³ Although strongly correlated with CHr, RET-He is an accurate estimate based on the scattered-light intensity of reticulocytes and the mean cellular hemoglobin.³ Reticulocyte hemoglobin equivalent is a slightly more common method of analysis because it can be performed on a Sysmex automated hematological analyzer.⁷

Reticulocyte hemoglobin content is not affected by the confounding factors that limit the efficacy of other markers of iron deficiency anemia. It is solely affected by parts of the hemoglobin synthesis pathway, specifically iron availability.³ Additionally, it provides information about hemoglobin earlier than other studies because it can detect hemoglobin content almost immediately after hematopoiesis.¹ Although iron deficiency may not produce technical anemia in mature erythrocytes for weeks, reticulocyte hemoglobin content can display subclinical iron deficiency by uncovering iron deficiencies that are functionally significant but not yet manifested in traditional iron indices.³

Clinical Applications of Reticulocyte Hemoglobin Content

As an initial management tool, CHr can be utilized as an early detector of iron status. The convenience for both physicians and patients is a key advantage of this laboratory value, especially for early identification of a disease process. One study demonstrated that reduced iron stores in female athletes created changes in reticulocytes first. As the iron deficiency intensified, the changes were then seen in erythrocyte indices. Furthermore, the frequency of latent iron deficiency in these females was high. This indicates a need for early screening, detection, and ultimately prevention of progression to anemia.⁶ Providers may be able to detect subclinical iron deficiency using CHr in those at high risk, ultimately preventing onset of anemia and reducing health care resources for prodromal symptoms.

Another important consideration is the implications of this tool in screening special populations, such as neonates. Neonates with iron deficiency are known to be at increased risk for motor and cognitive deficits.³ With the ability of this marker to detect inadequate iron in a fetal brain before true anemia manifests, the effects of iron deficiency anemia could be prevented rather than treated.

Although current studies have focused on screening, there is much to be explored in the treatment of iron deficiency anemia and the consequential response to therapy. For instance, CHr is directly proportionate to the severity of iron deficiency.⁸ This is of particular importance for treatment options. In patients with iron deficiency anemia, an increase in reticulocyte hemoglobin directly displays the most recent changes in hemoglobin, which specifically correlates to iron stores. In a patient who is receiving iron repletion therapy, the first physiologic aspect affected will be the reticulocytes. Two days after intravenous iron injections, changes can be seen in CHr.³ This could allow treatments to be adapted and adjusted earlier on in therapy, leading to reduced iron dosing, reduced costs, and possibly even reduced hospital stays.³ Many studies have demonstrated that CHr is an advantageous marker of iron repletion as it evaluates response to iron therapy before the traditional iron indices change or before positive clinical outcomes emerge.⁸

Another potential application of the reticulocyte hemoglobin measurement is the differentiation of hematologic conditions. Both iron deficiency anemia and beta thalassemia trait present similarly. Both conditions are displayed with microcytic hypochromic erythrocytes on blood smear. Usual diagnosis of thalassemia includes complete blood counts and hemoglobin electrophoresis with possible genetic testing. Recently, research has shown that CHr levels and the percentage of microcytic reticulocytes are significantly lower in patients with beta thalassemia than in those with iron deficiency.³ This differentiation is simpler with CHr testing than the traditional assessments for beta-thalassemia, thus allowing for more precise and earlier identification and treatment of these patients. Research continues to be conducted on using CHr in a new index or formula for detecting beta-thalassemia trait.³

Specific patient populations like infants and conditions including pregnancy, chronic kidney disease, hypothyroidism, and frequent blood donors are strongly associated with iron deficiency anemia. The value of CHr diagnostic and therapeutic marker can be seen in patients with anemia from chronic kidney disease. Many patients with chronic kidney disease display erythropoietin deficiency from kidney damage, however proper treatment may be hindered by an overlying iron deficiency.¹ Measuring ferritin cannot be relied upon to evaluate for iron deficiency, therefore measuring CHr is superior. RET-He or CHr can isolate decreased hemoglobin content in reticulocytes outside the context of confounding factors allowing for a functional iron deficiency to be corrected.¹ Once iron deficiency is addressed, further treatment for kidney disease can proceed unhindered.

Conclusion

With all of these promising applications, is it worthwhile for primary care providers to invest in technology to assess reticulocyte hemoglobin content? RET-He can be used in clinical applications and is cost-effective. A simple peripheral blood sample in an ethylenediaminetetraacetic acid tube is needed with no additional reagents. With the same tube of blood, analysis of CHr can be evaluated in an automatic processor alongside complete blood cell counts.⁷ With these instruments, RET-He can be measured rapidly, usually in less than 2 minutes.⁷ Although CHr requires a more specialized blood analyzer with certain reagents, it is more cost-effective compared with those required for serum ferritin measurement.³

As with all tools in the medical field, there is difficulty in normalizing its use. Sensitivity and specificity of most research regarding CHr varies widely with changes in the boundaries of what is considered anemic vs nonanemic.³ Great potential exists, however, for use of this marker in daily practice — from screening to diagnosis to treatment.

Charity Sorensen, PA-C, is a physician assistant practicing in primary care; Rachel Ziganti, MPA, PA-C, is a physician assistant working with the Department of Rheumatic and Immunologic Diseases of the Cleveland Clinic in Cleveland, Ohio.

References

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This article originally appeared on Clinical Advisor