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Reticulocyte Count and Reticulocyte Hemoglobin Content

Sections Reticulocyte Count and Reticulocyte Hemoglobin Content
Reference Range
Interpretation
Collection and Panels
Background
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References

Reference Range

The reticulocyte count is used to estimate the degree of effective erythropoiesis,^[1]which can be reported as absolute reticulocyte count or as a reticulocyte percentage.

The reference ranges for the reticulocyte count are as follows^[2]:

Adult/elderly/child: 0.5-2%
Infant: 0.5-3.1%
Newborn: 2.5-6.5%

Note that the reference ranges for automated reticulocyte count (absolute reticulocyte count), immature reticulocyte fraction (IRF), and reticulocyte specific hemoglobin content (mean reticulocyte hemoglobin content [CHr] and reticulocyte hemoglobin equivalent [Ret-He]) vary owing to the different methods and different instruments used. Each laboratory should determine reference values according to their own methods and instruments. A comparison of different reference ranges as reported by different authors can be found in a review published article by Piva et al.^[3]

Reticulocyte percentage versus absolute reticulocyte count

The reticulocyte count is used to estimate the degree of effective erythropoiesis,^[1]which can be reported as absolute reticulocyte count or as a reticulocyte percentage. In the latter case, if anemia is present, the reticulocyte percentage is spuriously high and may not reflect true bone marrow responses to anemia; therefore, the value has to be adjusted to a corrected reticulocyte percentage based on the patient’s hematocrit.^{[4, 5, 1, 6]}Using an automated hematology analyzer, the automated absolute reticulocyte count is reported.

Just as hematocrit is used to correct the reticulocyte percentage, the red blood cell (RBC) count is already incorporated in a similar purpose to generate the absolute reticulocyte count; therefore, it is not necessary to calculate a correction for absolute reticulocyte count.^[3]However, even when the degree of anemia is corrected for, another factor that can result in spuriously elevated reticulocyte percentage and absolute reticulocyte count is the time reticulocytes spend in the blood circulation prior to maturation.

In general, reticulocytes mature within one day of being released from bone marrow; however, in the setting of stress erythropoiesis, as in a high erythropoietin level (eg, in persons with severe anemia), reticulocytes are prematurely released from bone marrow to the blood circulation, increasing the number of days that reticulocytes stay in the blood circulation (maturation time of reticulocytes in days) and resulting in a spuriously high reticulocyte count.

The reticulocyte index, or reticulocyte production index, is a calculation that helps to alleviate the effect of the premature release of reticulocytes by taking into account maturation time of reticulocytes (ie, correction factor), in addition to correcting for the degree of anemia.^{[1, 3, 6]}

The absolute reticulocyte count and corrected reticulocyte percentage, as a marker of RBC production, provide an initial evaluation as to whether anemia is due to loss of RBCs or inadequate production.^{[5, 6]}In appropriate responses to peripheral RBC loss (bleeding or hemolysis), increased RBC production from the bone marrow is expected, and reticulocytosis (increased reticulocytes) should be apparent. On the other hand, the failure of the adequate bone marrow response to anemia usually correlates with a low reticulocyte count and is associated with bone marrow–related disorders.

Increased reticulocyte count reflects ongoing or recent RBC production activity, which may result from the following:^[7]

Post bleeding (trauma, gastrointestinal bleeding, menorrhagia)
Post hemolysis (hemolytic anemia, hemolytic disease of the newborn)
Response to therapy (iron supplementation, vitamin B-12 or folic acid supplementation, erythropoietin supplementation, bone marrow recovery following chemotherapy or bone marrow transplantation)

A decreased reticulocyte count reflects decreased RBC production, which may result from the following:^[7]

Vitamin B-12, folic acid, and iron deficiency (megaloblastic anemia, pernicious anemia, iron deficiency anemia)
Decreased erythropoietin level (chronic renal failure)
Aplastic anemia or bone marrow failure syndromes
Post radiation therapy
Bone marrow replacement by benign (metabolic storage diseases, infection, sarcoidosis) or malignant processes (leukemias, involvement by lymphomas or metastatic tumors)

Reticulocyte index (also known as the reticulocyte production index)

The reticulocyte index, or reticulocyte production index (RPI), which is corrected or adjusted for both premature release of reticulocytes from the bone marrow and the degree of anemia, is another parameter that provides an assessment for adequate bone marrow response to anemia.

An increased RPI (RPI >3) can be seen in the following scenarios:^[3]

Hemolytic anemias
Recent hemorrhage
Marrow response to therapy

A decreased RPI (RPI < 2) can be seen in the following:^[3]

Hypoproliferative disorder (ie, aplastic anemia)
Ineffective erythropoiesis, as seen in megaloblastic anemia

Immature reticulocyte fraction

Some automated hematology analyzers report an immature reticulocyte fraction (IRF), which provides information similar to the reticulocyte index.^[3]IRF is a quantitative measurement of the RNA content of the reticulocytes. Immature (younger) reticulocytes contain a higher RNA content than more mature reticulocytes. An increase in reticulocytes with the highest RNA content (increased IRF), therefore, reflects early marrow recovery from the conditioning regiments of stem cell transplantation, cancer chemotherapy, or treatment for nutritional anemias, which usually precedes the increase in absolute reticulocyte count.^[8]IRF has also been used to evaluate ineffective erythropoiesis and to differentiate megaloblastic anemia or myelodysplasia (increased IRF) from other causes.^{[4, 9]}

IRF can also been used with the reticulocyte count to narrow the causes of anemia. For example, a low absolute reticulocyte count with low IRF is associated with severe aplastic anemia or renal failure, while reticulocytopenia with a high IRF indicates repopulating marrow. Reticulocytosis with a high IRF is typically seen in acute hemolysis or blood loss, while a low to normal absolute reticulocyte count and high IRF is associated with dyserythropoiesis, as seen in an early response to iron therapy.^[10]

Measurement of reticulocyte-specific hemoglobin content

The automated hematology analyzer can also report a measurement of reticulocyte-specific hemoglobin content as mean reticulocyte hemoglobin content (CHr) or reticulocyte hemoglobin equivalent (Ret-He), depending on the type of instrument used. CHr and Ret-He, two comparable but not identical parameters, give a snapshot of the functional iron available for incorporation into hemoglobin within RBCs over the previous 3-4 days.^[10]A decreased value generally reflects reduced cellular hemoglobin content and is reliable in identifying functional iron deficiency. Furthermore, this parameter is the strongest predictor of iron deficiency anemia in children.^{[11, 12, 13]}

Estimating the reticulocyte-specific hemoglobin content (CHr or Ret-HE) is useful in the following scenarios:^[4]

Detecting functional iron deficiency in complex clinical settings, such as chronic inflammation and chronic renal disease, since serum ferritin can be falsely elevated as an acute-phase reactant despite low body iron storage, combined with the physiologic variation of serum iron and total iron-binding capacity (the conventional iron panel is therefore less useful in these situations)
May be a better predictor of marrow iron stores than traditional serum iron parameters in nonmacrocytic subjects
A more sensitive predictor of iron deficiency than hemoglobin for screening infants and adolescents for iron deficiency prior to the development of anemia

Collection and Panels

Specimen: Whole blood

Collection: EDTA containing Vacutainer tube (purple/lavender top; see image below)

Vacutainer tube, lavender top.

Panels: Reticulocyte count, or complete blood cell (CBC) count with reticulocyte count

Description

The absolute reticulocyte count is the number of reticulocytes in a volume of blood,^[4]which is a marker of RBC production.^[5]Reticulocytes are immature, but already anucleated, erythroid cells (RBCs) with residual detectable amounts of RNA, thereby capable of producing hemoglobin despite anucleation.^[14]RBCs are produced and mature in the bone marrow, and the majority are released in a fully mature form. In healthy adult individuals, a small proportion of the circulating RBCs are reticulocytes, representing the immediately preceding maturation state just before the fully developed RBCs.

The reticulocyte count provides an indirect insight into the bone marrow condition by distinguishing if the anemia is related to inadequate RBC production or accelerated loss/destruction. If the patient is anemic, the reticulocyte count percentage is falsely increased, in which case the value then needs to be adjusted based on the patient's hematocrit to the corrected reticulocyte count. This parameter, however, does not take into account premature release of reticulocytes from the bone marrow, which results in a longer time the reticulocytes spend in the circulation and a subsequent spuriously high reticulocyte count.

The reticulocyte production index is a factor that is adjusted for both the degree of anemia and the maturation time of reticulocytes (see calculation below).

The corrected reticulocyte percentage is calculated with the formula below.^{[1, 6]}

Corrected reticulocyte percentage = reticulocyte percentage x [actual hematocrit/normal hematocrit (usually 45)]

The reticulocyte index, or reticulocyte production index, is calculated with the formula below.^{[1, 6]}

Reticulocyte index = corrected reticulocyte percentage/maturation time in peripheral blood in days (correction factor)*

*Note: For simplicity, an average factor of 2 is often used; however, the factor is based on the patient's degree of anemia (hematocrit); see below

Hematocrits and corresponding correction factors are as follows:

Hematocrit, 45: Factor of 1
Hematocrit, 35: Factor of 1.5
Hematocrit, 25: Factor of 2
Hematocrit, 15: Factor of 2.5

Immature reticulocyte fraction

Immature reticulocyte fraction (IRF) is defined as the least mature fraction of reticulocytes and serves as a mean of assessing reticulocyte fraction.^[3]The maturity of reticulocytes is classified based on the amount of stained RNA content by automated machine using fluorescence. The most immature reticulocytes, produced when erythropoietin levels are high, contain more RNA content and fluoresce more strongly than the more mature ones normally present in the peripheral blood.^{[3, 10]}IRF can be used to help classify the cause of anemia, to assess for effective erythropoiesis, and to evaluate for marrow recovery.^{[10, 4]}

Reticulocyte-specific hemoglobin content

The mean reticulocyte hemoglobin content (CHr) and reticulocyte hemoglobin equivalent (Ret-He) are two equivalent parameters that capture the amount of hemoglobin available to the reticulocytes within the previous 3-4 days. Both parameters directly correlate with the functional availability of iron in the bone marrow.^{[15, 11]}

Indications/Applications

The reticulocyte count is not usually a part of a standard CBC count, but is ordered and used along with CBC, as CBC with reticulocyte count, to guide anemia workup or response to treatment. Depending on the type of automated hematology analyzer used in the laboratory, reticulocyte-specific hemoglobin content and IRF may be reported along with reticulocyte count to provide additional valuable information. These parameters are useful in the following situations:

Anemia workup (peripheral destruction vs failure of production)
Response to therapy (iron, vitamin B-12, folic acid supplementation)
Bone marrow recovery after bone marrow transplantation or intensive chemotherapy

Considerations

Smokers usually have an increased RBC count with an increased reticulocyte count (secondary polycythemia).^[7]

The reticulocyte count/percentage temporarily increases during the adaptation period to higher altitudes (temporary decrease in the reticulocyte count/percentage upon the adaptation to lower altitudes).^[7]

The reticulocyte count may be temporarily increased during pregnancy as the adaptation to the increased blood volume.

While their previous research indicated that earlier preterm infants have higher reticulocyte counts, Takahashi et al found in another study that preterm infants who are small for gestational age (SGA) have a lower reticulocyte count than do preterm infants who are not SGA. According to the investigators, this may suggest that SGA preterm infants adapt well during the early postnatal period to the extra-uterine environment.^[16]

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