What is the difference between reticulocyte count and absolute reticulocyte count?

What Happens in a Reticulocyte Count Test?

When you get this test, a lab tech will take a sample of blood from one of your veins.

In earlier years, doctors would put a drop of blood on a microscope slide and count the number of reticulocytes themselves. Today, machines calculate the results of nearly all reticulocyte count tests.

Why You Might Get One

A reticulocyte count test is often done when someone is believed to have an illness called anemia, which happens when your body doesn’t make enough red blood cells. That can leave you feeling weak and tired, short of breath, or having headaches and chest pain.

A retic count is often a follow-up to what’s known as a complete blood count or CBC. Most of the time, the CBC is the first test doctors use to diagnose anemia.

There are many different kinds of anemia. If your complete blood count suggests you have anemia, a reticulocyte count is one of several tests that can help tell your doctor which type:

• Aplastic anemia: Your reticulocyte count is low. That tells your doctor your bone marrow isn’t making red blood cells fast enough.
• Hemolytic anemia: Your reticulocyte count is high. This type of anemia destroys red blood cells before they would normally die, so your bone marrow has to work overtime to replace them.
• Iron deficiency anemia: A low reticulocyte count also can be a sign of this. It happens when your body doesn’t have enough iron to make red blood cells.
• Pernicious anemia: Your body doesn’t get enough vitamin B12, also producing a low reticulocyte count.

Other Reasons to Get One

A reticulocyte count test is also useful for people with sickle cell disease. That’s a disorder that makes your body produce red blood cells that are shaped like a crescent, or sickle, instead of being round.

Sickle cells die early and can caught in blood vessels, forming obstructions that cut off circulation to parts of the body. They can cause a form of anemia, because there aren’t enough healthy red blood cells to carry oxygen, as well as other painful or life-threatening illnesses that can put you in the hospital. A high reticulocyte count in someone with sickle cell disease suggests increased hemolysis, and points to a sickle cell crisis. Sickle cell crisis is usually painful and can be life-threatening.

Reticulocytes appear as polychromatophilic cells seen on a Wright- or Wright-Giemsa-stained blood film.

New methylene blue or brilliant cresyl blue are mixed with several drops of blood and incubated for 10 minutes in a tube before making a blood film. Reticulocytes are counted out of 1000 RBCs in a blood film and expressed as percentage of the RBCs. In cases of anemia, the number will be altered, so reticulocyte number per µl is calculated as RBCs/µl×% reticulocytes.

Reticulocytes are increased as the bone marrow responds to anemia. A reticulocyte number of less than 10 000 per µl is considered to represent no or minimal regenerative response; 10 000–60 000 per µl is a poor regenerative response; 60 000–200 000 per µl is considered a moderate response; and 200 000–500 000 reticulocytes per µl is a maximal regenerative response.

Reference ranges for erythrocyte parameters in the laboratory animals covered in this volume are provided in Table 3.9.

Table 3.9. Erythrocyte Values1

RBC Count (3106/µl)Hct/PCV (%)Hgb conc (g/dl)RBC Lifespan (days)RBC diam (microns)MCV (m3)MCH (pg/cell)MCHC (g/dl)Reticulocytes (%)ReferencesRabbit4.9–7.831–5010–17.4575.0–7.857.5–7517.1–23.928.2–371.7–6.3Thrall et al., 2004Brewer, 2006Melillo, 2007Vennen and Mitchell, 2009Guinea pig4–1130–5011–17N/A6.6–7.970–9523–2725–400–6.1Thrall et al., 1974Riggs, 2009Hamster2.7–12.330–5910–19.250–785–764–7820–2628–37N/AThrall et al., 2004Heatley and Harris, 2009Gerbil7–1035–5210–17.910N/A46.6–6016.1–19.430.6–33.3N/ADonnelly and Quimby, 2002Heatley and Harris, 2009Chinchilla5–1030–559–15N/AN/A32.1–69.210.4–19.820–38.5N/ADonnelly and Quimby, 2002Riggs and Mitchell, 2009Deer mouse10–1238–5212–16N/AN/A36–4611–1530–35N/ASealander, 1960, 1961Gough and Kilgore, 1964Meagher, 1998Kangaroo rat7–947–5315–17N/AN/A57.3–66.018.6–2530–35N/AScelza and Knoll, 1982Intress and Best, 1990Cotton ratN/AN/A13–14N/AN/AN/AN/AN/AN/ADolyak and Leone, 1953

N/A=values not available.

Reticulocyte count

The reticulocyte count serves as an important tool to assess the bone marrow’s ability to increase RBC production in response to an anemia. Reticulocytes are young RBCs that lack a nucleus but still contain residual ribonucleic acid (RNA) to complete the production of hemoglobin. Normally they circulate peripherally for only 1 day while completing their development. The adult reference interval for the reticulocyte count is 0.5% to 2.5% expressed as a percentage of the total number of RBCs.13 The newborn reference interval is 1.5% to 6.0%, but these values change to approximately those of an adult within a few weeks after birth.13 An absolute reticulocyte count is determined by multiplying the percent reticulocytes by the RBC count (Table 16.1). The reference interval for the absolute reticulocyte count is 20 to 115 × 109/L, based on an adult RBC count within the reference interval (inside front cover).4,7 A patient with a severe anemia may seem to be producing increased numbers of reticulocytes if only the percentage is considered. For example, an adult patient with 1.5 × 1012/L RBCs and 3% reticulocytes has an absolute reticulocyte count of 45 × 109/L. The percentage of reticulocytes exceeds the reference interval, but the absolute reticulocyte count is within the reference interval. For the degree of anemia, however, both these results are inappropriately low. In other words, production of reticulocytes within the reference interval is inadequate to compensate for an RBC count that is approximately one-third of normal.

Two successive corrections are made to the reticulocyte count to obtain a better representation of RBC production. First, to obtain a corrected reticulocyte count, one corrects for the degree of anemia by multiplying the reticulocyte percentage by the patient’s hematocrit and dividing the result by 45 (the average normal hematocrit). If the reticulocytes are released prematurely from the bone marrow and remain in the circulation 2 to 3 days (instead of 1 day), the corrected reticulocyte count must be divided by maturation time to determine the reticulocyte production index (RPI) (Table 16.1). The RPI is a better indication of the rate of RBC production than is the corrected reticulocyte count.4 The reticulocyte count and derivation of RPI is discussed in Chapter 11.

In addition, state-of-the-art automated blood cell analyzers determine the fraction of immature reticulocytes among the total circulating reticulocytes, called the immature reticulocyte fraction (IRF). The IRF is helpful in assessing early bone marrow response after treatment for anemia and is covered in Chapter 12.

Analysis of the reticulocyte count plays a crucial role in determining whether an anemia is due to an RBC production defect or to premature hemolysis and shortened survival defect. If there is shortened RBC survival, as in the hemolytic anemias, the bone marrow tries to compensate by increasing RBC production to release more reticulocytes into the peripheral circulation. Although an increased reticulocyte count is a hallmark of the hemolytic anemias, it can also be observed after acute blood loss (Chapter 20).4,7,8 Chronic blood loss, on the other hand, does not lead to an appropriate increase in the reticulocyte count, but rather leads to iron deficiency and a subsequent low reticulocyte count. Thus an inappropriately low reticulocyte count results from decreased production of normal RBCs, as a result of either insufficient or ineffective erythropoiesis.

Automated reticulocyte counting

Reticulocyte counting is the last of the manual cell-counting procedures to be automated and has been a primary focus of hematology analyzer advancement in recent years. The imprecision and inaccuracy in manual reticulocyte counting are due to multiple factors, including stain variability, slide distribution error, statistical sampling error, and interobserver error.51 All these potential errors, with the possible exception of stain variability, are correctable with automated reticulocyte counting. Increasing the number of RBCs counted produces increased precision.52 This was evidenced in the 1993 College of American Pathologists pilot reticulocyte proficiency survey (Set RT-A, Sample RT-01) on which the CV for the reported manual results was 35% compared with 8.3% for results obtained using flow cytometry.53 Precision of automated methods has continued to improve. The manual reticulocyte results for one specimen in the 2000 Reticulocyte Survey Set RT/RT2-A showed a CV of 28.7%, whereas the CV was 2.8% for results obtained using one of the automated methods.54 Automated reticulocyte analyzers may count 32,000 RBCs compared with 1000 cells in the routine manual procedure.55

Available automated reticulocyte analyzers include flow cytometry systems such as the FACS system from Becton, Dickinson or the Coulter EPICS system; the Sysmex R-3500, R-500, XE-2100, XE-5000, and XN-series systems; the CELL-DYN 3500R, 3700, and 4000 systems; the Coulter LH 750 systems and the UniCel DxH800; and the Siemens ADVIA 2120, 2120i, and 120. All these analyzers evaluate reticulocytes based on optical scatter or fluorescence after the RBCs are treated with fluorescent dyes or nucleic acid stains to stain residual RNA in the reticulocytes. Because neither the FACS nor EPICS system is generally available in the routine hematology laboratory, the discussion here is limited to the other analyzers.

The Sysmex R-3000/3500 is a stand-alone reticulocyte analyzer that uses auramine O, a supravital fluorescent dye, and measures forward scatter and side fluorescence as the cells, in a sheath-stream, pass through a flow cell by an argon laser. The signals are plotted on a scattergram with forward scatter intensity, which correlates with volume, plotted against fluorescence intensity, which is proportional to RNA content. Automatic discrimination separates the populations into mature RBCs and reticulocytes. The reticulocytes fall into low-fluorescence, middle-fluorescence, or high-fluorescence regions, with the less mature reticulocytes showing higher fluorescence. The immature reticulocyte fraction (IRF) is the sum of the middle-fluorescence and high-fluorescence ratios and indicates the ratio of immature reticulocytes to total reticulocytes in a specimen. The XE-5000, the XT-2000i, and the XN series also determine the reticulocyte count and IRF by measuring forward scatter and side fluorescence. They also have a parameter called RET-He (reticulocyte hemoglobin equivalent) that measures the hemoglobin content of the reticulocytes.56 It uses a proprietary polymethine dye to fluorescently stain the reticulocyte nucleic acids. This is similar to the reticulocyte hemoglobin content (CHr) parameter on the ADVIA 2120i (discussed later). Platelets, which also are counted, fall below a lower discriminator line.57 The Sysmex SE-9500/9000 + RAM-1 module uses the same flow cytometry methodology for reticulocyte counting as the R-3500.16 Offline specimen preparation is not required. The smaller Sysmex R-500 uses flow cytometry with a semiconductor laser as the light source and polymethine supravital fluorescent dye to provide automated reticulocyte counts.28

The CELL-DYN 3500R performs reticulocyte analysis by measuring 10-degree and 90-degree scatter in the optical channel (MAPSS technology) after the cells have been isovolumetrically sphered to eliminate optical orientation noise. The RBCs are stained with the thiazine dye new methylene blue N in an off-line sample preparation before the specimen is introduced to the instrument. The operator simply must change computer functions on the instrument before aspiration of the reticulocyte preparation.46 The CELL-DYN Sapphire also uses MAPSS technology but adds fluorescence detection to allow fully automated, random access reticulocyte testing.24,47 The RBCs are stained with a proprietary membrane-permeable fluorescent dye (CD4K530) that binds stoichiometrically to nucleic acid and emits green light as the cells, in a sheath-stream, pass through a flow cell by an argon ion laser. Platelets and reticulocytes are separated based on intensity of green fluorescence (scatter measured at 7 degrees and 90 degrees), and the reticulocyte count along with the IRF is determined.47

Beckman Coulter also has incorporated reticulocyte methods into its primary cell-counting instruments: LH 700 series systems and the UniCel DxH800. The Coulter method uses a new methylene blue stain and the VCS technology described earlier. Volume is plotted against light scatter (DF 5 scatterplot) and against conductivity (DF 6 scatterplot), which correlates with opacity of the RBC. Stained reticulocytes show greater optical scatter and greater opacity than mature RBCs. Relative and absolute reticulocyte counts are reported, along with mean reticulocyte volume and maturation index or IRF.55

The Siemens ADVIA 2120, 2120i, and 120 systems enumerate reticulocytes in the same laser optics flow cell used in the RBC/platelet and BASO channels described earlier. The reticulocyte reagent isovolumetrically spheres the RBCs and stains the reticulocytes with oxazine 750, a nucleic acid–binding dye. Three detectors measure low-angle scatter (2 to 3 degrees), high-angle scatter (5 to 15 degrees), and absorbance simultaneously as the cells pass through the flow cell. Three cytograms are generated: high-angle scatter versus absorption, low-angle scatter versus high-angle scatter (Mie cytogram or RBC map), and volume versus hemoglobin concentration. The absorption cytogram allows separation and quantitation of reticulocytes, with additional subdivision into low-absorbing, medium-absorbing, and high-absorbing cells based on amount of staining. The sum of the medium-absorbing and high-absorbing cells reflects the IRF. Volume and hemoglobin concentration for each cell are derived from the RBC map by applying Mie scattering theory.26,58 Unique reticulocyte indices (MCVr, CHCMr, RDWr, HDWr, CHr, and CHDWr) are provided. The CHr or reticulocyte hemoglobin content of each cell is calculated as the product of the cell volume and the cell hemoglobin concentration. A single-parameter histogram of CHr is constructed, with a corresponding distribution width (CHDWr) calculated.22,23 These reticulocyte indices are not reported on the routine patient printout but are available to the operator. Figure 12.12 is a reticulocyte printout from an ADVIA 120, showing the cytograms and reticulocyte indices.

Automation of reticulocyte counting has allowed increased precision and accuracy and has greatly expanded the analysis of immature RBCs, providing new parameters and indices that may be useful in the diagnosis and treatment of anemias. The IRF, first introduced to indicate immature reticulocytes, shows an early indication of erythropoiesis. The IRF and the absolute reticulocyte count can be used to distinguish types of anemias. Anemias with increased marrow erythropoiesis, such as hemolytic anemia, have a high total reticulocyte count and increased IRF, whereas chronic renal disease has decreased absolute count and an IRF indicating decreased marrow erythropoiesis.58,59 An increased IRF and normal to decreased absolute reticulocyte count indicates an early response to therapy in nutritional anemias.59 Use of both tests is a reliable indicator of changes in erythropoietic activity and may prove to be a valuable therapeutic monitoring tool in patients.59 The reticulocyte maturity measurements also may be useful in evaluating bone marrow suppression during chemotherapy, monitoring hematopoietic regeneration after bone marrow or stem cell transplantation, monitoring renal transplant engraftment, and assessing efficacy of anemia therapy.58-62 The reticulocyte hemoglobin content, CHr (Advia) and Ret-He (Sysmex), provides an assessment of the availability of iron for erythropoiesis (Chapters 8 and 17). The additional reticulocyte indices derived on the ADVIA 2120 and 120 are valuable in following the response to erythropoietin therapy, and the CHr in particular has proved useful in the early detection and diagnosis of iron-deficient erythropoiesis in children.62,63 The National Kidney Foundation Kidney Disease Outcomes Quality Initiative recommends the addition of the reticulocyte hemoglobin content to the CBC, in addition to the reticulocyte count and ferritin level, to assess the iron status in patients with chronic kidney disease.64 Widespread use of the new parameters may be limited by the availability of instrumentation.

3.1.8 Reticulocyte Count

Reticulocytes are non-nucleated, immature RBCs formed in the blood marrow before being released in the blood. The reticulocyte count is used to estimate the degree of effective erythropoiesis and can help in the diagnosis of different types of anemia. A high reticulocyte count occurs due to increased EPO response which is seen in anemia due to hemolysis, blood loss, or response to treatment. A low reticulocyte count can occur due to decreased production of RBCs from reduced bone marrow response, nutritional deficiency (e.g., iron, vitamin B12, folate), or decreased EPO levels in chronic renal failure.

The reticulocyte count can be expressed either as a percentage of all RBCs, the absolute reticulocyte count, the corrected reticulocyte count, the reticulocyte production index (RPI) or as the immature reticulocyte fraction (IRF). In individuals with anemia, the percent of reticulocytes present in the blood may appear high compared to the overall number of RBCs. A calculation called the corrected reticulocyte count, also referred to as the reticulocyte index (RI), can be used to provide a more accurate assessment of bone marrow function. The RI is calculated as RI = Reticulocyte count (%) × [Measured hematocrit/Normal hematocrit], in which the patient's hematocrit is compared with a normal hematocrit value. The RPI can be used to correct for the degree of reticulocyte immaturity by factoring in how early the reticulocytes were released from the bone marrow and how long it will take for them to mature in the bloodstream. The RPI is calculated as RPI = (RI) × (1/maturation time), which varies with the hematocrit. Reticulocyte maturity can now be estimated based on the staining intensity of reticulocytes which is proportional to their RNA content. The IRF is a better indicator of bone marrow response than the total reticulocyte count in certain conditions such as post-bone marrow transplantation to demonstrate successful engraftment or to gauge adequacy of response to EPO therapy in patients with anemia associated with chronic renal failure [20,21]. It is calculated as the ratio of immature reticulocytes to the total number of reticulocytes. In healthy individuals, reticulocytes circulate in the peripheral blood for approximately 1–2 days after being released from the bone marrow before becoming RBCs. The reticulocyte lifespan can increase to 3 or more days as a result of premature release during periods of increased erythropoietic demand.

Reticulocyte count

The reticulocyte count is the most commonly used test to detect accelerated erythropoiesis and is expected to rise during hemolysis or hemorrhage. Assuming the bone marrow is healthy and there are adequate nutrients, all measures of reticuloycyte production should rise: absolute reticulocyte count, relative reticulocyte count, reticulocyte production index, and immature reticulocyte fraction. The association of reticulocytosis with hemolysis is so strong that if an anemic patient has an elevated reticulocyte count and hemorrhage is ruled out, a cause of hemolysis should be investigated. The reticulocyte increase usually correlates well with the severity of the hemolysis. Exceptions occur during aplastic crises of some hemolytic anemias and in some immunohemolytic anemias with hypoplastic marrow, which suggests that the autoantibodies were directed against the bone marrow erythroid precursors and circulating erythrocytes.32 Chapters 11 and 16 describe the interpretation of reticulocyte indices in patients with anemia.

Immature reticulocyte fraction

The IRF is the proportion of reticulocytes that have a particularly high RNA fraction. IRF is calculated as a ratio of immature reticulocytes to total reticulocytes. This parameter provides an early and sensitive index of marrow erythropoietic activity by measuring the most immature reticulocytes in the circulation. Increased immature reticulocytes indicate acceleration of RBC production.15 Schiza et al.,16 from Ioannina, Greece, reported IRF values from 181 infants of 32 to 36 weeks’ gestation, at 2 and 6 weeks and at 3, 6, 9, and 12 months. The IRF values were not part of an automated CBC. Values at 2 weeks were 29 ±9% in those born at 34 to 36 weeks’ gestation, with lower values of 22 ±9% in those born at 32 to 34 weeks (P <0.001). A decrease was seen in both groups by 3 months, to 13 ±6% and 12% ±5%, respectively. Our neonatal reference interval values (see Fig. 7.2) showed a similar pattern, falling from 36.2 ±5.6% at birth to 10.1 ±4.9% by 90 days.

4 Species Differences in Marrow Release

Reticulocyte maturation begins in the bone marrow and is completed in the peripheral blood and spleen in dogs, cats, and pigs. As reticulocytes mature, they lose the surface receptors needed to adhere to fibronectin and thrombospondin components of the extracellular matrix, presumably facilitating their release from the bone marrow (Telen, 2000). Residual adhesion molecule receptors on newly released reticulocytes may explain their tendency to concentrate in the reticular meshwork of the spleen (Patel et al., 1985).

Reticulocytes become progressively more deformable as they mature, a characteristic that also facilitates their release from the marrow (Waugh et al., 2001). To exit the extravascular space of the marrow, reticulocytes press against the abluminal surfaces of endothelial cells that make up the sinus wall. Cytoplasm thins and small pores (0.5 to 2 μm) develop in endothelial cells, which allow reticulocytes to be pushed through by a small pressure gradient across the sinus wall (Lichtman and Santillo, 1986; Waugh, 1991). Pores apparently close after cell passage.

Relatively immature aggregate-type reticulocytes are released from canine bone marrow; consequently, most of these cells appear polychromatophilic when viewed following routine blood film staining procedures (Laber et al., 1974). Absolute reticulocyte counts oscillate with a periodicity of approximately 14 days in some dogs, suggesting that canine erythropoiesis may have a homeostatically controlled physiological rhythm (Morley and Stohlman, 1969). Reticulocytes are normally not released from feline bone marrow until they mature to punctate-type reticulocytes; consequently, few or no aggregate reticulocytes (<0.4%), but up to 10% punctate reticulocytes, are found in blood from normal adult cats (Cramer and Lewis, 1972). The high percentage of punctate reticulocytes results from a long maturation time with delayed degradation of ribosomes (Fan et al., 1978). Reticulocytes are generally absent in peripheral blood of healthy adult cattle and goats, but a small number of punctate types (0.5%) may occur in adult sheep (Jain, 1986). Based on microscopic examination of blood films stained with new methylene blue, equine reticulocytes are absent from blood normally and rarely released in response to anemia. However, low numbers of reticulocytes have been reported in the blood of normal and anemic horses using an Advia 120 (Siemens Medical Solutions Diagnostics, Tarrytown, New York) automated analyzer (Cooper et al., 2005). Either the instrument is more sensitive than microscopic evaluation, or values reported in normal horses represent “noise” in the instrument.

Reticulocyte count (RETIC)

Reticulocytes are immature red blood cells containing residual RNA. Reticulocytes can be counted by instrumentation or by haemocytometer. Automated reticulocyte counts are performed similarly to automated RBCs. Before counting, red blood cells are stained with a dye that stains for nucleic acid (such as acridine orange) to differentiate reticulocytes from mature red blood cells. For manual reticulocyte counts, whole blood is mixed with a supravital dye such as new methylene blue and blood smears are prepared. The dye causes clumping and staining of residual nucleic acid present in immature cells. The stained cells (reticulocytes) are counted as a percentage of total red blood cells. The absolute reticulocyte count is determined by multiplying the total RBC by the percentage of reticulocytes. The number of circulating reticulocytes is higher in mice (200–500 × 109/L) than in most other species, due to the short lifespan of the mouse red blood cell. Units for reticulocyte count vary among laboratories, but generally are reported in the same units as red blood cells or in the same units as platelets (cells × 109/L).

Reticulocyte count

Reticulocytes are immature red cells, which are released from the bone marrow into the circulation one to two days before maturation. An increase in the reticulocyte count is indicative of an increase in red cell production (erythropoiesis) to meet physiological demand, for example in haemolytic anaemia.

Reticulocytes contain ribosomal RNA, which can be detected by two main methods. When unfixed red cells are incubated with dyes such as new methylene blue, the ribosomal RNA precipitates out and appears as a blue reticular network within the cells, which can be visualised using a light microscope, allowing the reticulocytes to be counted. Most automated FBC analysers now perform reticulocyte counts by flow cytometry, using a fluorescent dye that binds to the ribosomal RNA; the number of reticulocytes can be expressed as a percentage of total red cells and as an absolute count.

What is an absolute reticulocyte count?

What are the two types of reticulocytes?

What is another name for reticulocyte count?

What does a high reticulocyte absolute count mean?