What is reticulocyte count

Reticulocytes are newly produced, relatively immature red blood cells (RBCs). Reticulocytes are “young” red blood cells that are released by the bone marrow before they become fully mature. A reticulocyte count is used to determine the number and/or percentage of reticulocytes in your blood to help evaluate conditions that affect red blood cells, such as anemia or bone marrow disorders. Reticulocytes are newly produced, relatively immature red blood cells. They form and mature in the bone marrow before being released into the blood. Reticulocytes are the erythroid cells in the peripheral blood that are in a discrete, penultimate phase of maturation ¹. The nucleus has been removed, usually before the red cells enter the peripheral blood. However, some of the extranuclear RNA remains. This residual RNA generally is lost progressively during the 24 hours after the cell enters the circulation. Reticulocytes thus represent a distinctive cohort of cells, those most recently entering the peripheral blood ¹. Reticulocytes differ from other red blood cells in that they have a more convoluted shape, and are about 8% larger than the more mature cells ¹. These latter two distinctions are not so clear-cut as is the presence of residual RNA. With the typical Wright’s stain used for routine examination, only the earliest reticulocytes with the most residual RNA will be “polychromatophil” (i.e., more bluish than the mature erythrocytes).

Red blood cells are produced in the bone marrow, where blood-forming (hematopoietic) stem cells differentiate and develop, eventually forming reticulocytes and finally becoming mature red blood cells. Reticulocytes are approximately 24% higher in volume in comparison with mature red blood cells. Unlike most other cells in the body, mature red blood cells have no nucleus, but reticulocytes still have some remnant genetic material (RNA). As reticulocytes mature, they lose the last residual RNA and most are fully developed within one day of being released from the bone marrow into the blood. The reticulocyte count or percentage is a good indicator of the ability of a person’s bone marrow to adequately produce red blood cells (erythropoiesis).

Red blood cells typically survive for about 120 days in circulation, and the bone marrow must continually produce new red blood cells to replace those that age and degrade or are lost through bleeding. Normally, a stable number of red blood cells is maintained in the blood through continual replacement of degraded or lost red blood cells.

A variety of diseases and conditions can affect the production of new red blood cells and/or their survival, in addition to those conditions that may result in significant bleeding. These conditions may lead to a rise or drop in the number of red blood cells and may affect the reticulocyte count.

The reticulocyte count may be used:

• As a follow up to abnormal results on a complete blood count (CBC), red blood cell count, hemoglobin or hematocrit, to help determine the cause
• To determine if the bone marrow is functioning properly and responding adequately to the body’s need for red blood cells
• To help detect and distinguish between different types of anemia
• To monitor response to treatment, such as that for iron-deficiency anemia
• To monitor bone marrow function following treatments such as chemotherapy
• To monitor function following a bone marrow transplant

Most often, a reticulocyte count is performed with an automated instrument (hematology analyzer) and can be done simultaneously with a complete blood count (CBC), which includes an red blood cell count, hemoglobin and hematocrit. Either an absolute number of reticulocytes and/or a percentage of reticulocytes can be reported. For a percentage, the number of reticulocytes is compared to the total number of red blood cells:

• Reticulocyte (%) = [Number of Reticulocytes / Number of total Red Blood Cells] X 100

Several other tests may be used in conjunction with a reticulocyte count to further evaluate someone for a condition affecting red blood cell production. Some examples include:

• Iron studies
• Vitamin B12 and Folate
• Haptoglobin
• G6PD
• Erythropoietin

Sometimes a bone marrow aspiration and biopsy may be done in follow up to abnormal results on initial tests. This procedure is invasive and is not done on everyone. It can, however, provide additional information, if necessary.

Higher than normal percentage of reticulocytes: Acute or chronic bleeding (hemorrhage) or increased red blood cell destruction (hemolytic disorders) can lead to fewer red blood cells in the blood, resulting in anemia. Hemolytic disorders, whether intrinsic (e.g., hemoglobinopathy or enzymopathy) or extrinsic (e.g., traumatic, heart valve, acquired immune hemolytic anemia). As long as the hemolytic cause is not corrected, the reticulocyte count will remain elevated. A particular danger to patients with chronic hemolytic anemia is transient reticulocytopenia from a drug or infection. The body compensates for this loss or to treatment of deficiency anemias (such as iron deficiency anemia or pernicious anemia) by increasing the rate of red blood cell production and by releasing red blood cells sooner into the blood, before they become more mature. When this happens, the number and percentage of reticulocytes in the blood increases until a sufficient number of red blood cells replaces those that were lost or until the production capacity of the bone marrow is reached. In the normal subject, marrow hypoplasia for even a week makes little difference because a week’s erythropoiesis is only 7/120, or 6%, of the red cell mass. In contrast, the patient with chronic hemolytic anemia and a 20% reticulocyte percentage is making 20% of cells each day. If the cells continue to be destroyed prematurely, even a fall to half of the accustomed hyperproduction could be disastrous. Therefore, the patient with chronic hemolytic anemia (e.g., sickle cell anemia) should have interval reticulocyte counts as well as hemoglobin measured. To the extent that the hemolysis has caused a “shift” of erythropoiesis, the cells will be larger. Also, the more anemia caused by hemolysis, the greater the proportion of polychromatophilic cells. Therefore, an extremely high reticulocyte percentage may represent only a modest increase in absolute erythropoiesis. In these disorders, bone marrow precursors are not only increased in number; they also are altered in their maturation. This is analogous to the changes in platelet production in immune thrombocytopenia.

The reticulocyte percentage is also increased in the recovery from a nutritional anemia (e.g., iron) or the recovery of erythropoiesis after bone marrow suppression (e.g., chemotherapy). If the marrow is normal after the therapy, the degree of reticulocytosis will parallel the degree of anemia. A similar pattern is seen in the acute reticulocytosis following sudden blood loss. As the recovery progresses, the reticulocyte percentage will abate to normal. Usually in these acute marrow responses, there is no “shift” of erythropoiesis. The reticulocyte percentage and number are high, but the proportion of polychromatophilic cells is normal. The number of marrow erythroid cells is increased, but their maturation is normal. This represents a lesser, and perhaps qualitatively different, stimulation of the bone marrow. This is similar to reactive thrombocytosis.

Lower than normal percentage of reticulocytes: Decreased red blood cell production may occur when the bone marrow is not functioning normally. This can result from a bone marrow disorder such as aplastic anemia. Diminished production can also be due to other factors, for example, anemia of chronic disease, cirrhosis of the liver, kidney disease, radiation or chemotherapy treatments for cancer, a low level of the hormone erythropoietin, or deficiencies in certain nutrients such as iron, vitamin B12, or folate. Decreased production leads to fewer red blood cells in circulation, decreased hemoglobin and oxygen-carrying capacity, a lower hematocrit, and a reduced number of reticulocytes as old red blood cells are removed from the blood but not fully replaced.

To be certain that the reticulocyte percentage is abnormally low, a repeat measurement may be necessary because as few as four or five reticulocytes in the thousand cells counted (0.4 to 0.5%) may be within the duplicate error of “normal.” When the correction factors are used, the absolute reticulocyte count (percentage adjusted for degree of anemia) may be below normal, even though the percentage is normal. In such cases the bone marrow is not responding appropriately to the anemia and therefore is, in an absolute sense, hypoproliferative. This approach helps to distinguish physiologically those red cell disorders in which the number and/or proliferative activity of the bone marrow erythroid precursors is deficient. However, in practice, because of the limitations described above, there will be a broad “gray zone” in which it is unclear whether the reticulocyte percentage (or count) is normal or not. Hypoplastic thrombocytopenia has a similar basis.

Occasionally, both the reticulocyte count and the red blood cell count will be increased because of excess red blood cell production by the bone marrow. This may be due to an increased production of erythropoietin, disorders that cause chronic overproduction of red blood cells (polycythemia vera), and cigarette smoking.

Some drugs may increase or decrease reticulocyte counts.

Normal reticulocyte count

It is assumed that the normal red cell life span is 120 days and that the duration of a reticulocyte in the peripheral blood is 1 day. It follows that reticulocytes at a random time, in a normal subject at a steady state, will be 1/120, or 0.8% of all red cells ². Allowing for a 10% variation in red cell life span would theoretically give a range of 0.7 to 0.9%. However, there is considerable statistical variability when the number of observations is small. Further, there is observer variability in deciding what is positively stained (a minimum of 25% intraobserver and 50% interobserver variation). Therefore, the duplicate error in a given reticulocyte percentage approaches 100% (our normal subjects” reticulocyte percentage varied by an average of 80% among five samples over 2 months). Because some laboratories are more liberal than others about designating cells as reticulocytes, the clinician must know the local normal values and range, and be certain that these represent the laboratory’s actual experience rather than values given in a reference book. Our range of normal values is 0.4 to 3.0%.

The reticulocyte percentage in the peripheral blood is an indication of the rapidity of red cell turnover if the patient is in a steady state. However, the number of reticulocytes released into the blood reflects the amount of erythropoiesis on a given day. The absolute number of reticulocytes is determined as the reticulocyte index, or “corrected” reticulocyte count. To determine this, the reticulocyte percentage is adjusted by the ratio of the observed hematocrit to the expected hematocrit, yielding an absolute number of reticulocytes. Sometimes a “second correction” is made. If, subjectively judged, there is pronounced polychromatophilia in the peripheral blood film, it is assumed that younger reticulocytes are disproportionately numerous. Such younger reticulocytes will retain their RNA longer than average, and so they represent a cohort of cells that will be perceived as reticulocytes for longer than 1 day. Their persistence as reticulocytes would give a falsely high percentage of cells as representing a single day’s red cell production. The magnitude of the “second correction” tends to parallel the first. For instance, if the reticulocyte count is 10.0% and the hematocrit is 25%, and polychromasia is seen on the slide:

It is assumed that the normal red cell life span is 120 days and that the duration of a reticulocyte in the peripheral blood is 1 day. It follows that reticulocytes at a random time, in a normal subject at a steady state, will be 1/120, or 0.8% of all red cells. Allowing for a 10% variation in red cell life span would theoretically give a range of 0.7 to 0.9%. However, there is considerable statistical variability when the number of observations is small. Further, there is observer variability in deciding what is positively stained (a minimum of 25% intraobserver and 50% interobserver variation). Therefore, the duplicate error in a given reticulocyte percentage approaches 100% (our normal subjects” reticulocyte percentage varied by an average of 80% among five samples over 2 months). Because some laboratories are more liberal than others about designating cells as reticulocytes, the clinician must know the local normal values and range, and be certain that these represent the laboratory’s actual experience rather than values given in a reference book. Our range of normal values is 0.4 to 3.0%.

The reticulocyte percentage in the peripheral blood is an indication of the rapidity of red cell turnover if the patient is in a steady state. However, the number of reticulocytes released into the blood reflects the amount of erythropoiesis on a given day. The absolute number of reticulocytes is determined as the reticulocyte index, or “corrected” reticulocyte count. To determine this, the reticulocyte percentage is adjusted by the ratio of the observed hematocrit to the expected hematocrit, yielding an absolute number of reticulocytes. Sometimes a “second correction” is made. If, subjectively judged, there is pronounced polychromatophilia in the peripheral blood film, it is assumed that younger reticulocytes are disproportionately numerous. Such younger reticulocytes will retain their RNA longer than average, and so they represent a cohort of cells that will be perceived as reticulocytes for longer than 1 day. Their persistence as reticulocytes would give a falsely high percentage of cells as representing a single day’s red cell production. The magnitude of the “second correction” tends to parallel the first. For instance, if the reticulocyte count is 10.0% and the hematocrit is 25%, and polychromasia.

Reticulocytes are said to be large enough to cause an increase in mean cell volume (MCV) if the percentage is markedly elevated. This occurs in only a small group of subjects with very high reticulocyte percentages—those with chronic immune hemolytic anemia. These latter patients are thought to have a “shift” of erythropoiesis, in which a cell division is skipped and cells larger than normal are produced. Otherwise, reticulocytes are only about 8% larger than the red cells into which they mature. Therefore, only in autoimmune hemolytic anemia should increased red cell size (MCV) be attributed to the reticulocytosis.

A new technique for reticulocyte counting is use of flow cytometry. This approach uses a dye that selectively stains RNA, and distinguishes reticulocytes from other RNA-containing cells by another variable such as size. This technique has not yet been widely used, but is under active study at several centers.

What is corrected reticulocyte count or reticulocyte index?

When an individual has anemia (the number of red blood cells (RBCs), hemoglobin and hematocrit are low), the percentage of reticulocytes may appear high compared to the overall number of red blood cells. This is in part due to the immature cells’ early release from the bone marrow into circulation and the longer time they spend maturing in the blood (from the normal 1 day to 3 or 4 days). In order to get a more accurate assessment of bone marrow function, the reticulocyte percentage (%) is often corrected with a calculation called a corrected reticulocyte count (CRC) or a reticulocyte index (RI). This calculation compares the person’s hematocrit with a normal hematocrit value:

• Reticulocyte Index or Corrected Reticulocyte Count = Reticulocyte count (%) X [Measured hematocrit / Normal hematocrit]

What is a reticulocyte production index?

In addition to the corrected reticulocyte count or reticulocyte index (RI), a calculation called the reticulocyte production index (RPI) is sometimes calculated to correct for the degree of reticulocyte immaturity, reflecting how early the reticulocytes were released from the bone marrow and how long it will take them to mature in the bloodstream. The reticulocyte production index (RPI) and maturation time vary with the hematocrit.

• Reticulocyte Production Index = (Reticulocyte Index) X (1/maturation time)

What is immature reticulocyte fraction?

The immature reticulocyte fraction (IRF) is calculated as a ratio of immature reticulocytes to the total number of reticulocytes. It is reported by an automated blood analyzer when a reticulocyte count test is performed. In certain conditions, immature reticulocyte fraction (IRF) is a better indicator of bone marrow response than a total reticulocyte count.

Will a blood transfusion affect reticulocyte results?

Yes. Your healthcare provider will determine how long you should wait after a transfusion before having a reticulocyte count performed.

How else might my doctor evaluate the cell production in my bone marrow?

In some cases, a procedure called a bone marrow aspiration may be performed to obtain a sample of marrow to evaluate under the microscope. Sometimes this is the best way for a healthcare provider to determine how well the bone marrow is functioning.

When is reticulocyte count ordered?

A reticulocyte count may be ordered when:

• Complete blood count (CBC) results show a decreased red blood cell count and/or a decreased hemoglobin and hematocrit
• A healthcare practitioner wants to evaluate bone marrow function
• An individual has signs and symptoms of anemia or chronic bleeding, such as paleness, lack of energy, fatigue, weakness, shortness of breath, and/or blood in the stool
• A person has been diagnosed and is being treated for a condition known to affect red blood cell production, such as iron deficiency anemia, vitamin B12 or folate deficiency, or kidney disease (which can affect the production of erythropoietin, a hormone produced by the kidneys that stimulates red blood cell production by the bone marrow)
• An individual is undergoing radiation or chemotherapy
• A person has received a bone marrow transplant
• Occasionally when a person has an increased number of red blood cells and elevated hemoglobin and hematocrit, to help determine the degree and rate of overproduction of red blood cells

What does the reticulocyte count result mean?

Reticulocyte count results must be interpreted carefully and in conjunction with results of other tests, such as a red blood cell count, hemoglobin (Hb), hematocrit (Hct) or complete blood count (CBC). In general, the reticulocyte count (absolute number or percentage) is a reflection of recent bone marrow activity. Results may indicate whether a disease or condition is present that is generating an increased demand for new red blood cells and whether the bone marrow is able to respond to the extra requirement. Occasionally, results may indicate overproduction of red blood cells.

A low reticulocyte percentage or count reflects a marrow unable to compensate for anemia. A high reticulocyte percentage or count reflects a marrow that is attempting to compensate for red cell destruction, or recovering from anemia. In between the two clear-cut extremes are those subjects who have reticulocyte percentages within the normal range, including the technologic error. These normal reticulocyte disorders include many cases of nutritional disorders (iron, folate, vitamin B12 deficiency); the confusing spectrum of chronic liver disease and alcohol abuse; leukemias and other marrow infiltrative disorders; and nonanemic hemoglobinopathies such as hemoglobin AS or AC. The differential is long and usually other, more specific tests will be needed to establish a diagnosis in patients with normal reticulocyte values. Adjustment for hematocrit or polychromasia usually is valuable only in cases of marked anemia.

When anemia is present (i.e., low red blood cells, low hemoglobin, low hematocrit) and the bone marrow is responding appropriately to the demand for increased numbers of red blood cells, then the bone marrow will produce more and allow for the early release of more immature red blood cells, increasing the number of reticulocytes in the blood.

A high reticulocyte count with low red blood cells, low hemoglobin, and low hematocrit (anemia) may indicate conditions such as:

• Bleeding: If an individual bleeds (hemorrhage), then the number of reticulocytes will rise a few days later in an attempt to compensate for the red cell loss. If someone has chronic blood loss, then the number of reticulocytes will stay at an increased level as the marrow tries to keep up with the demand for new red blood cells (although it may not be high if the blood loss leads to iron deficiency).
• Hemolytic anemia: In this condition, anemia is caused by increased destruction of red blood cells. The bone marrow increases red blood cell production to compensate, resulting in a high reticulocyte count.
• Hemolytic disease of the newborn: This condition causes increased red blood cell destruction, similar to hemolytic anemia described above.

A low reticulocyte count with low red blood cells, low hemoglobin, and low hematocrit (anemia) may be seen, for example, with:

• Iron deficiency anemia
• Pernicious anemia or folic acid deficiency
• Aplastic anemia
• Radiation therapy
• Bone marrow failure caused by infection or cancer
• Severe kidney disease; this may cause a low level of erythropoietin.
• Alcoholism
• Endocrine disease

When an individual has anemia, the percent of reticulocytes present in the blood may appear high compared to the overall number of red blood cells. In order to get a more accurate assessment of bone marrow function, a calculation called a corrected reticulocyte count or reticulocyte index (RI) may be reported. The reticulocyte index (corrected reticulocyte count) is calculated by comparing the person’s hematocrit with a normal hematocrit value. Other calculations that may also be reported include the reticulocyte production index (RPI) and an immature reticulocyte fraction (IRF). The immature reticulocyte fraction was previously referred to as the reticulocyte maturity index; however, immature reticulocyte fraction is now the most common term used to quantify the younger fraction of reticulocytes.

The reticulocyte test gives an indication of the presence of a disease or condition but is not directly diagnostic of any one particular disease. It is a sign that further investigation may be necessary and a tool that can be used to monitor the effectiveness of therapy.

If reticulocyte numbers rise following chemotherapy, a bone marrow transplant, or treatment of an iron or vitamin B12 or folate deficiency, then bone marrow red blood cell production is beginning to recover.

When an individual does not have anemia, or has a high red blood cell count (polycythemia), a high reticulocyte count may indicate an overproduction of red blood cells. Some conditions that may cause this include:

• Polycythemia vera
• Tumor that produces excess erythropoietin

People who move to higher altitudes may have high reticulocyte counts for a while as their body adapts to the lower oxygen content of their new location. Smokers also may demonstrate an increased number of red blood cells and reticulocytes.

Reticulocyte counts may be high during pregnancy. Newborns have a higher percentage of reticulocytes, but the number drops to near adult levels within a few weeks of birth.

Traditionally, reticulocyte counts have been done manually by looking at a specially stained slide under the microscope and counting the number of reticulocytes in a number of fields of view. Although it is used occasionally, the manual method has been replaced by automated methods that allow for a greater number of cells to be counted, thus enhancing the accuracy of reticulocyte counts. The automated method is considered to be more accurate in identifying reticulocytes.

References

1. Bessman JD. Reticulocytes. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 156. Available from: https://www.ncbi.nlm.nih.gov/books/NBK264
2. Bessman JD. Reticulocytes. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 156. Available from: https://www.ncbi.nlm.nih.gov/books/NBK264/