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Reticulocytes are immature red blood cells (RBCs) that is seen in the bone marrow after through nuclear extrusion from the orthochromatic normoblasts. They are released into the peripheral blood as mature RBCs, after completion of maturation in the bone marrow. The reticulocyte count reflects the erythropoietic activity of the bone marrow, the rate of reticulocyte delivery from the bone marrow into the peripheral blood, and the rate of reticulocyte maturation. Reticulocyte enumeration is also of value in monitoring bone marrow regenerative activity after chemotherapy or bone marrow transplantation. Manual counting of reticulocytes by light microscopy with supravital dyes for RNA remains the standard method of reticulocyte enumeration. However, automated methods of reticulocyte enumeration developed during the past decade are much more accurate, precise, and cost-effective than manual counting, and are increasingly being performed in the clinical laboratory. The differentiation of the reticulocyte is based on the presence of RNA. The newer techniques provide a variety of reticulocyte related parameters, such as the reticulocyte maturation index and immature reticulocyte fraction, which are not available with light microscopy. These new parameters are under evaluation in the clinical diagnosis and monitoring of hematological disorders.
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1. Introduction
Reticulocytes are immature red blood cells (RBCs) produced in the bone marrow and released into the peripheral blood where the terminal maturation into RBCs occurs for next couple of days. Any alteration in reticulocyte count is an indicator of active or failed erythropoiesis, in response to anemias or other causes of bone marrow dysfunction [1].
The first description of reticulocytes was made in 1865 by Wilhelm Heinrich Erb, a German neurologist when he discovered the population of granulated erythrocytes while observing the effect of acetic and picric acid on the development of erythrocytes. He erroneously regarded these cells as transitional forms between leucocytes and erythrocytes. Ehrlich described the stained material as fine, dense, and elegant networks as a feature of senescent erythrocytes rather than of young ones [2]. The reticular substance was first regarded as a degenerative material, a “coagulation necrosis” or a substance produced by the action of certain deleterious agents on corpuscles.
Theobald Smith brilliantly asserted that reticulocytes represented young red cells. Further investigators designated these cells as erythrocytes with “substantia granulo-filamentosa,” the Americans used such terms as “reticulated red cell” or “vital-staining erythrocyte.”
In 1922, Edward Bell Krumbhaar, first coined the term “reticulocyte” when he stated that: “Erythrocytes revealing granular filamentous substance by the methods of vital staining may be conveniently designated ‘reticulocytes” [3].
Ludwig Heilmeyer, a German internist, proposed the still well-known classification of reticulocytes maturity in 1932 (Figure 1). In 1944, Pierre Dustin, a Belgian pathologist, showed that granular “reticulated” substance is RNA [5]. In 1947, Giovanni Astaldi, an Italian hematologist, classified reticulocytes into 3 stages of maturity, as in the current maturation classification used in flow cytometry. In 1918, it was demonstrated that reticulocytes are able to synthesize hemoglobin and absorb iron [6].
Figure 1.
Maturation stages of reticulocytes according to Heilmeyer classification [4].
Over the years acridine orange, a fluorescent dye, replaced the use supravital stain, improving the sensitivity of manual microscopic count of reticulocytes [7].
The “reticulocyte” term has derived from the reticulum of RNA and protein precipitated by the fixation and staining that seen microscopically after supravital staining [8, 9]. These supravital granules represent precipitated rough endoplasmic reticulum along with polyribosomes. Thus, the human reticulocytes are defined as mature red cells that form a reticulum network or granules on exposure to supravital stains, such as new methylene blue or brilliant cresyl blue [10].
Although this definition has been revised several times, in 1997 it was chosen to be the universal standard by the Clinical and Laboratory Standards Institute and the International Council for Standardization in Hematology (NCCLS-ICSH) [11].
2.2 Morphology
Romanowsky type stain, such as May-Gru¨nwald Giemsa or Wright stain, causes the RNA to disappear during alcohol fixation and reticulocytes acquire slightly larger size than mature erythrocytes, with a uniform polychromatic blue-gray color. On staining with new methylene blue (NMB) or brilliant cresyl blue (BCB), the RNA precipitates and becomes visible with the characteristic scattered granules under the microscope.
Reticulocytes must have at least 2 blue staining granules, visible without fine microscope adjustment and present away from the cell margin, as standard morphological definition provided by NCCLS-ICSH, in 1997 [11]. These granules can mimic Heinz bodies.
2.3 Properties of reticulocytes
Reticulocytes in comparison with mature erythrocytes, have following characteristics [12]:
The early reticulocyte contains mitochondria, a small number of ribosomes, the centriole, and remnants of Golgi bodies.
Early reticulocytes continue to synthesize hemoglobin, with approximately 20–30% of the total hemoglobin of the RBC.
Reticulocytes are larger (8.5 μ) than mature erythrocytes and they gradually decrease in size during maturation.
The volume of reticulocytes and mature erythrocytes increases in pathological conditions but the ratio of their volumes remain constant: approximately 1.24
Reticulocytes are less dense than mature RBCs, due to lower Hb concentration and higher water content of reticulocytes.
When the orthochromatic normoblast (late stage of erythropoiesis) loses its nucleus, it becomes a reticulocyte that persist in the bone marrow for next few days and is subsequently released into the circulation for terminal maturation [13, 14].
Maturation of reticulocyte is a continuous process with various morphologic, biochemical, and functional changes that lead to remodeling of membrane, changes in volume, and elimination of membrane-bound organelles and ribosomes [15]. Immature or early reticulocytes are biochemically more active than mature ones with intact cellular functions of Hb production and absorption of iron [16]. Circulating reticulocytes are unable to synthesize Hb and cannot further increase their Hb content.
Maturation of reticulocytes is a complex sequential mechanism of enucleation, caused by condensation of chromatin, vesicular trafficking, and selective autophagy [17]. The ultimate maturation occurs when the basophilic reticular filamentous substance in the reticulocyte disappears [18]. Intracytoplasmic organelles such as the mitochondria, ribosomes, and endosomal vesicles are eliminated by a mitochondrial death program which includes physiologic events of macroautophagy and mitoptosis [19, 20].
Early reticulocyte maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, and β1 integrin) through the endosome exosome pathway. In contrast, late maturation is characterized by the generation of large glycophorin A coated vesicles of autophagic origin [21, 22].
Recent studies have suggested that the small amount of RNA that remains in reticulocytes might still be essential for reticulocyte maturation to form normal biconcave erythrocytes [23].
During the maturational remodeling of the membrane cytoskeleton, by vesiculation and endocytosis, reticulocytes lose about 24% of their volume and surface area, and increase their stability and deformability [24].
As already mentioned, a series of progressive physiological and biochemical changes occur during the differentiation of reticulocytes into mature RBCs [25]. The most important of these changes include:
Synthesis of hemoglobin and its cytoplasmic accumulation.
Loss of protein-synthesizing apparatus and mitochondria.
Condensation and contraction of chromatin to cause extrusion of the nucleus.
Exosome formation to cause loss of cell-surface membrane receptor expression.
Alteration in cholesterol and phospholipid levels of cell membrane.
Changes in intracytoplasmic enzyme levels of glucose-6-phosphate dehydrogenase (G-6-PD).
A number of classification systems have been attempted based on the maturation of reticulocytes along with their morphology. Maturity classification is based on the quantity of the granular/reticular filamentous substance and its distribution in their cytoplasm.
The first attempt was made by Heilmeyer and Westharer, who divided the cells into 4 groups (Groups I-IV), designated by Roman numerals and characterized by a progressive reduction in the compactness of the reticulum. Therefore, reticulocyte maturation predominantly assesses the relative proportion of mRNA (see Figure 1).
Group 0 – nucleated RBCs with a dense perinuclear reticulum.
Group I – most immature reticulocytes with extruded nuclei having a dense, coherent mass of RNA.
Group II – reticulocytes with extensive but loose reticular network.
Group III – reticulocytes with scattered reticular network.
Group IV – most mature reticulocytes with scattered remnants of RNA.
According to Lowenstein, in steady-state erythropoiesis the circulating reticulocytes are more than 60% in Group IV, 30% in Group III, and less than 1% in Groups I or II.
4.1 Stress Reticulocytosis
Some specific hematological disturbances (e.g. severe anemia or hemolysis) are compensated with accelerated erythropoiesis (called stress erythropoiesis), which causes release of the immature, larger and more stained form of reticulocytes into the peripheral blood, which are called “stress reticulocytes” and appear as polychromatophilic cells on Romanowsky staining. All polychromatophils are reticulocytes on a Wright-stained blood smear. Reticulocyte production can increase up to 20 folds above the base line values of 1 to 2 million reticulocytes per second when stressed with intense hematopoiesis. This increased production is accomplished by increased production and shortening of marrow maturation time [26, 27]. Stress reticulocytosis is seen in bone marrow regeneration following autoimmune hemolysis, chemotherapy induced anemia, administration of therapy in nutritional anemia, and use of erythropoiesis-stimulating agents (ESAs). Stress reticulocytes are multilobular and motile, in contrast to the cup-shaped non-motile mature reticulocytes.
Till the end of last century, the standard method of counting reticulocytes was based on the visual detection of RNA ribosomal networks (reticulum), by microscopic examination of supravitally stained peripheral blood smears. But, this method has several sources of imprecision in the manual microscopic counting of reticulocytes, including different staining with variation, distributional variability for quality of blood film, intra and inter-observer variations, and inadequate number of cells counted. As per NCCLS-ICSH guidelines, supravital staining with New Methylene Blue (NMB) still remains the recommended method for optimal agreement/correlation assessments. NMB staining procedures have been standardized to improve the accuracy and reliability of reticulocyte manual counting [28].
Reticulocyte counting should be done within 6 hours if the sample is kept at room temperature, or up to 72 hours if the blood sample is refrigerated at 2-6°C.
5.1 Manual methods
5.1.1 Photographic methods
In 1960s photographs used to be obtained on wet preparation from fresh oxalated blood mixed with brilliant cresyl blue in isotonic saline. 30–35 fields containing reticulocytes used to be printed on glossy paper with final magnification of approximately 4000x.
5.1.2 Planimetric method
Reticulocytes stained with brilliant cresyl blue wet preparations are photographed and the resultant Kodachromes are projected onto butcher paper. Then, pairs of reticulocytes and adjacent RBCs are circled and traced with a planimeter.
5.1.3 Light microscopic method
Equal volumes of anticoagulated (preferably EDTA) peripheral whole blood and supravital stains are mixed and incubated for at least 20 minutes. A thin smear of the stained blood preparation is made on a microscope slide, a counterstain (usually Wright’s) is applied, and the slide is examined by light microscopy. An adequate number of RBCs (minimum 1000 for optimal analytical precision) in a well-stained area are examined, and the proportion of reticulocytes is determined. Reticulocytes possess a blue granular precipitate, which can vary from individual small blue granules to a network of blue reticular material. Reticulocytes have a faint, diffuse basophilic hue termed as polychromasia (Figure 2).
Figure 2.
Photomicrograph of peripheral blood smears stained with new methylene blue, 400x. Reticulocytes (arrowhead) are differentiated from mature RBCs (arrow) by the presence of an intracellular granular precipitate [29].
The reticulocyte count is usually reported as a percentage of total RBC count. The normal mean percentage reticulocyte count by NMB light microscopy is 1.0% to 3.0%. In cases of anemia, this relative reticulocyte count is misleading because reduced RBC count causes erroneous elevation in reticulocyte count. Under these circumstances, reticulocyte count is corrected with respect to patient’s packed cell volume (PCV) as Reticulocyte index to compensate the decrease in mature RBCs. The PCV corrects the percentage reticulocyte count to the baseline PCV i.e. 0.45 using the following formula:
The normal ARC is between 50,000 and 150,000 reticulocytes/mL (5× 1010 and 1.5 × 1012 reticulocytes/L).
Manual microscopic RC is highly subjective and tedious, which results in high level of imprecision, with a coefficient of variations of up to 50% [30]. This imprecision in manual counting of reticulocytes can be attributed to:
Different supravital stains (NMB or BCB)
Staining variations
Quality of blood film
Intra and interobserver variation
Number of reticulocytes and RBCs counted
Stain precipitates and cellular interferences (platelets, leucocytes fragments)
The College of American Pathologists and National Committee for Clinical Laboratory Standards (NCCLS) has defined reticulocytes as the cell containing two or more precipitate granules not attached to cytoplasmic membrane following staining by new methylene blue and the use of Miller ocular disks, to standardize the microscopic counting and reduce the imprecision.
Hence, clinical serial assessment of erythropoietic activity of bone marrow in patients receiving myelotoxic or hematinic therapies by this manual counting of reticulocytes is impractical due to poor precision and methodologic limitations.
5.2 Immunofluorescence method
First attempt of reticulocyte enumeration by using immunofluorescence microscope was done in 1950s by Kozenow and Mai. They used RNA specific fluorochrome dye i.e., acridine orange. Subsequently, different fluorochromes used by others for reticulocyte enumeration later on like pyronin Y, thioflavin T, DiOCl, proflavin. Reticulocyte enumeration by this method gives spuriously high count sometimes due to some interference present in the blood sample such as platelet clumps, nucleated RBCs, Howell-Jolly bodies, Heinz bodies, Pappenheimer bodies, Basophilic stippling and Malarial parasites.
5.3 Fluorescence microscopy
The prefixed whole blood in formalin is mixed with a dilute, buffered solution of the dye and counted as a wet preparation. Ultraviolet to blue light is required for the excitation of these dyes, and a green long-pass filter must be placed for detection of the fluorescence emission.
5.4 Conventional flow cytometry
Thioflavin T analogue has been widely used as specific fluorochrome for reticulocyte enumeration to improve counting accuracy and made flow cytometric reticulocyte enumeration practical as TO excitation occurs in the visible region of the spectrum. These flow cytometers rely on the enumeration of reticulocytes using nucleic acid binding fluorescent dyes and measurement of fluorescence emission. Reticulocytes are identified and enumerated within the gated RBC population on the basis of fluorescence intensity. The quantitation of the intensity of green fluorescence reveals young, immature reticulocytes as brightly fluorescent (high RNA content), while maturing reticulocytes show an intermediate fluorescence intensity (intermediate RNA content), and older reticulocytes show dim fluorescence (low RNA content).
5.5 Automated reticulocyte instrumentation
All automated instruments for reticulocyte enumeration are capable of rapid analysis of whole blood sample in flow through systems, with the red cell population interrogated on a cell-by-cell basis by laser light. Now-a-days the reticulocyte counting is fully integrated into the automated complete blood count (CBC) in high-throughput hematology analyzers. The reticulocyte RNA content is detected by fluorochrome Auramine O, while the cell size is determined by forward light scattering using an argon ion laser as the light source. Automated flow cytometric analysis has replaced traditional microscopic quantitation of reticulocytes.
5.6 Reticulocyte-specific monoclonal antibodies
Reticulocyte analysis with fluorochrome-labeled monoclonal antibodies against reticulocyte surface receptors is available only for research studies of the reticulocyte and diseases of the erythron. Fluorochrome-labeled monoclonal antibodies specific for different reticulocyte antigens have been evaluated as reagents for reticulocyte enumeration. The antigens presently evaluated include the CD71 molecule (transferrin receptor, TfR, and T9 antigen), CD36, and other antigens. CD71 receptors are abundant on the surface of early reticulocytes, but gradually decrease during reticulocyte maturation, and are absent from mature RBCs. CD36 and the integrin α4-β1 complex are expressed on reticulocytes but not mature erythrocytes. CD36-positive reticulocytes correlate with the stress reticulocyte fraction in patients with chronic hemolytic diseases.
The recent development in automation of reticulocyte counts provide accurate RC with enhanced precision along with reliable and accurate measurements of RNA content and cellular indices such as mean reticulocyte volume, mean reticulocyte Hb concentration, and its content. These novel parameters have been studied with prompt interest for their clinical usefulness and the utility of reporting these analytes with their appropriate interpretation.
During intense erythropoietic stimulation, immature, large and basophilic precursor macroreticulocytes (“shift cells”) from the bone marrow are released prematurely into the peripheral blood. This causes a shortened reticulocyte maturation time in the bone marrow (depends on the severity of stress erythropoieisis), and the longer reticulocyte maturation time in the peripheral blood. Since these shift cells have a cell volume about 25% larger than that of normal cells, a correction for RBC maturation time and the PCV must be done when they comprise more than 5% of the total reticulocytes [31]. The correction is referred as “shift correction” or “reticulocyte production index”, and calculated by the following formula (Figure 3).
Figure 3.
The relationship between PCV (hematocrit) and blood maturation time. The total RBC maturation time is approximately 4.5 days. During normal erythropoieisis 3.5 days of maturation occurs in bone marrow and last one day in peripheral blood. However, in anemia the marrow maturation time progressively shortens, and reticulocytes circulate for a correspondingly longer period of time in the peripheral blood to compensate the degree of anemia [32].
RPI was proposed to correct the reticulocyte percentage for peripheral blood maturation time based on the hematocrit value. It helps to alleviate the effect of the premature release of reticulocytes by taking into account maturation time of reticulocytes. Thus RC is corrected or adjusted for both premature release of reticulocytes and the degree of anemia. Stressed erythropoiesis is accomplished by increased production and shortening of the fraction of time that reticulocytes mature in marrow and proportionally prolongs their maturation time in circulation, thus increasing reticulocyte circulation time. RPI is used for evaluating erythropoiesis and classifying anemias. RPI is normally between 2 and 3.
RPI of less than 2 suggests for hypo-proliferative erythropoiesis and more than 3 is applied for hypo-proliferative state of erythropoiesis.
Clinical interpretation of RPI
Increased RPI (RPI >3)
Hemolytic anemias (Autoimmune)
Recent hemorrhage
Marrow response to therapy in nutritional anemia (EPO)
Different populations of reticulocytes depend on the content of RNA in the enucleated cytoplasm. This RNA content can be assessed by the different intensity signals of fluorescence or light scattering/absorbance obtained on the flow cytometric based automated hematoanalyzers. The software-based algorithm in the autoanalyzers discriminate these reticulocytes into three areas of clusters according to stain intensity. High-fluorescence light scatter/absorbance reticulocytes correspond to young or immature reticulocytes, whereas maturing reticulocytes have medium-fluorescence light scatter, and older reticulocytes have low fluorescence light scatter. These three classes are called high-fluorescence ratio (HFR), medium high-fluorescence ratio (MFR), and low-fluorescence ratio (LFR), respectively.
The RMI is directly proportional to the amount of reticulocyte intracellular RNA. Maturational stages of reticulocytes in peripheral blood depends upon the level of anemia, disease state and iron status. Thalassemia, megaloblastic anemia, anemia of uremia and myelodysplastic syndrome (MDS) are associated with delayed reticulocyte maturation. Hence, RMI is used as adjunct parameter in the evaluation of hematological disorders and therapeutic monitoring of erythropoietic activity. Responsive marrow is expected to manifest a high RMI along with a subnormal RC.
Immature reticulocyte fraction is better accepted term internationally to quantify the younger fraction of reticulocyte as a sum of HFR and MFR, to avoid the ambiguity in the interpretation of different fluorescence intensities of RMI. It is expressed as a fraction (0.00–1.00). IRF assesses reticulocyte maturation by the intensity of the staining that reflects the mRNA content. Assessment of IRF is clinically useful when it is evaluated in correlation with absolute RC.
Clinical utility of IRF:
Early marker of engraftment in bone marrow- IRF increases very early when the bone marrow engraftment is successful and it precedes other parameters, like absolute neutrophil count (ANC), reticulated platelets, and RC. Increase in IRF occurs up to 1 week sooner than increase in ANC. IRF value greater than 10% indicates early marrow recovery and more than 20% from the post BMT value suggests successful erythroid engraftment. Hence, serial determination of IRF after bone marrow transplantation (BMT) is used to demonstrate successful engraftment.
Early marker for stem cell mobilization- IRF has been proposed as an early marker of CD34+ mobilization for peripheral stem cell harvesting to optimize the timing for stem cell collection following growth factor mobilization or cytotoxic drug therapy.
Effective monitoring of therapeutic efficacy of erythropoietin therapy- IRF increases in response to treatment with ESAs before there is an increase of RC in renal failure, AIDS, and MDS. Thus, indicate adequate erythropoietic stimulation.
Monitoring for efficacy of anemia treatment- As the IRF increases earlier than the reticulocyte number, it is useful in monitoring the efficacy of therapy in nutritional anemias. In ineffective erythropoiesis, IRF is increased while reticulocyte count is reduced or normal, in some cases of MDS or in dyserythropoietic anemia.
Screening of Hereditary Spherocytosis: High RC without proportionate elevation in IRF can be suspicious for HS. A RC/IRF ratio higher than 7.7 can be used as a cut off for the screening of all HS cases as a diagnostic algorithm [33].
The clinical utility of IRF has also been reported in a variety of conditions, like: the monitoring of anemia treatment and neonatal transfusion needs; monitor response to EPO in a blood conservation program; renal transplant engraftment from Epo production; the detection of occult or compensated hemorrhages or hemolysis, and aplastic crisis in hemolytic anemias; and the early diagnosis and monitoring of aplastic anemias (Table 1).
10. Reticulocyte mean cell Hemoglobin content (CHr)
As understood the early reticulocytes continue to synthesize hemoglobin, with approximately one fourth of the total hemoglobin of the RBC. Hence, CHr is the measurement of the Hb content of reticulocytes expressed in pg./cell. CHr is the product of the cellular volume and the cellular Hb concentration. The measurement of CHr can directly reflect the functional availability of iron in that time frame of life span of reticulocyte (up to 4 days) and is helpful in real-time assessment of the functional state of erythropoiesis. It can be of help in identifying iron deficiency before the development of IDA and also its serum level can guide for the route of administration of iron supplementation. Critically low value of CHr warrants for intravenous iron therapy, because oral iron is ineffective in preventing iron-deficient erythropoiesis. The prediction of the absence of bone marrow iron stores can better be diagnosed by CHr less than 28 pg., over than MCV, serum ferritin or transferrin saturation. Adequate iron levels must be ensured to optimize Hb production in a balance with erythropoietic stimulation by Epo in cases of chronic renal disease and it can be assessed by CHr.
11. Summary
The final stage of RBC differentiation occurs in the peripheral blood. The reticulocytes that are released by the bone marrow still contain RNA. On the way of maturation reticulocytes gradually lose their rough endoplasmic reticulum and mitochondria to become mature RBC after about 3–4 days in the peripheral blood. Since the number and characteristics of the reticulocytes in the peripheral blood gives insight about the activity of the bone marrow, reticulocyte counting has become fundamental part of the hematopoietic evaluation now-a-days. Circulating reticulocytes decrease in patients with impaired bone marrow function, and increase in cases of hemolysis with normal bone marrow activity. Reticulocyte enumeration by light microscopy, with the use of a supravital dye (viz. NMB), which binds to the RNA in the reticulocyte still remains the standard method for RC. However, the accuracy and precision of this assay are greatly compromised by its subjective nature. In contrast, automated techniques of reticulocyte enumeration are more precise, accurate, objective, and cost-effective. A variety of RNA-specific fluorescent dyes have been utilized for automated reticulocyte enumeration, and some hematology analyzers utilize optical light scatter analysis to perform reticulocyte analysis on specimens stained with NMB or other dyes. In addition to relative and absolute reticulocyte counts, automated techniques provide information regarding the age distribution of reticulocytes in form of the RMI, IRF, CHr. There is extensive evidence that these parameters are useful in the accurate classification of anemia patients, and monitoring patients receiving EPO or recovering from chemotherapy or bone marrow transplantation. The recent trend to incorporate reticulocyte analysis into the routine hematology analyzer has made automated reticulocyte analysis increasingly such common that, perhaps in recent future, the manual reticulocyte count will become an obsolete technique.
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Written By
Ashish Kumar Gupta and Shashi Bhushan Kumar
Submitted: 30 June 2022Reviewed: 16 August 2022Published: 09 October 2022
Open access peer-reviewed chapter
