Postscript

Introduction

• Approximately 2 million new red cells, called reticulocytes, are produced each second in adults.
• These cells emerge from the erythroblast population of the bone marrow without their nuclei but with a near complete complement of hemoglobin. 
• Reticulocytes:
  • Are formed in the bone marrow when an orthochromatic erythroblast ejects its nucleus.
  • Mature in the bone marrow for 2-3 days before their release into the circulation.
  • Complete their transformation into mature red blood cells over the next 24 hours in the circulation. 
• Because red blood cells have a long lifespan of 120 days, it is difficult to detect recent changes in erythropoiesis based on an analysis of the circulating mass of red blood cells, most of which were produced weeks-months earlier. Reticulocyte counts provide a window into recent erythropoietic activity. 
• The term reticulocyte was derived from the deep blue precipitate seen in these cells after staining with new methylene blue and other supravital dyes that bind and cross-link RNA and aggregate other organelles (precipitation of rough endoplasmic reticulum with associated polyribosomes).
• Reticulocyte counting has long been used in an attempt to:
  • Gain more immediate insight into the level of erythropoietic activity.
  • Determine whether the response is appropriate to the degree of anaemia:
    • Decreased reticulocyte count indicates bone marrow underproduction (inappropriate response).
    • Increased reticulocyte count indicates bone marrow erythropoiesis (appropriate response). 
  • Evaluate a patient’s response to therapeutic intervention, for example nutritional supplements in iron, folate, or vitamin B, deficiency states. 
  • Assess the cause for an elevated mean cell volume (MCV) (reticulocytes have an increased MCV).

Definitions

Term	Definition	Comment
Reticulocyte	Stage of erythrocyte development following enucleation of the orthochromatic normoblast still containing measurable RNA. When stained with supravital dyes, the remaining mRNA and ribosomes give the red cells a “reticular” mesh-like network, hence the name “reticulocyte”.	Reticulocytes are an index of bone marrow hemopoietic activity and are usually increased in hemolysis and in response to bleeding.
Polychromatophilic cells	Immature non-nucleated red cells with a blue hue in their cytoplasm; the blue may be barely visible or may be marked. Polychromatic red cells are typically larger than normal red cells. They tend to lack central pallor.	All polychromatophils are reticulocytes; however, not all reticulocytes are polychromatophils on a Wright-stained blood smear.
Stress reticulocytes	Very young reticulocytes released into the peripheral blood in response to a severely anemic state.	Found in the circulation only under conditions erythropoietic stress and are not present in nonanemic individuals.
Vital staining	A stain that brings fresh blood in contact with the stain before the cells are altered either by heat, alcohol or fixative.	Examples include methylene blue and crystal violet.
Manual reticulocyte analysis	Reticulocytes enumerated using vital stain and manual counting under a microscope.	See section below for disadvantages.
Automated reticulocyte analysis	Reticulocytes enumerated using automated fluorescent flow cytometry.	Uses RNA-specific fluorescent dyes (e.g. thiazole orange or polymethine dye) which binds to cytoplasmic RNA in the reticulocytes.
Reticulocyte %	Number of reticulocytes relative to total number of red blood cells.	The normal mean percentage reticulocyte count is 1.0% to 1.5%, with 3% being the upper limit of normal.
Reticulocyte index (RI)	A reticulocyte count corrected for anemia.	Also called the corrected reticulocyte count (CRC). RI <2% with anemia indicates decreased production of reticulocytes. RI >3% with anemia indicates appropriate marrow response to anemia.
Bone marrow responsiveness index (BMRI)	Calculated as the absolute reticulocyte count x patient’s Hb/normal Hb.	This index with a cutoff value of <121 is able to discriminate hemolysis with concomitant inadequate reticulocytopenia with a sensitivity of 90% and specificity of 65% in 205 patients with congenital dyserythropoietic anemia type II.
Reticulocyte production index (RPI)	A reticulocyte count corrected for both anemia (reticulocyte index) and delayed maturation time in peripheral blood with various levels of anemia.	Also known as reticulocyte maturation index; RPI of more than 3 indicates an appropriate bone marrow response (regenerative anemia), while an RPI of less than 2 indicates an inadequate compensatory response to correct the anemia (arregenerative anemia).
Absolute reticulocyte count	The % reticulocytes x total red blood cell count.	A more accurate method to correct for the effect of anemia; does not correct for maturation time.

About reticulocytes

• A reticulocyte is formed when an bone marrow orthochromatic erythroblast ejects its nucleus.
• The early reticulocyte contains:
  • Mitochondria
  • Small number of ribosomes
  • The centriole
  • Remnants of Golgi bodies.
• Reticulocytes, in comparison with mature red blood cells, show:
  • Higher volume (by about 25%; 115 fL compared to 85 fL)
  • Greater diameter (10–15 mm compared to 6–8 mm)
  • Lower Hb concentration (by about 17%; 27 g/dL compared to 33 g/dL)
• After about 2 days in the bone marrow, reticulocytes are normally released into the peripheral blood and undergo final maturation over the next 24 hours:
  • Although these cells have lost most of their protein synthetic capabilities they retain a residuum of RNA. The polysomal remnants take on the appearance of a reticulum when stained supravitally.
  • The maturation is complete when the basophilic filamentous substance that characterizes the reticulocyte disappears.
• As reticulocytes mature:
  • Hemoglobin synthesis gradually decreases.
  • Cellular organelles are progressively lost.
  • Cell volume decreases through:
    • Loss of membranes and intracellular remnants due to
      exosome/vesicle formation/shedding.
    • Actions of transporters that regulate the cation/anion and water content and thereby cell volume.
  • Their shape changes from larger, mobile, and irregular cells to discoid cells that are biconcave, smaller, and more uniform.
• Although the term reticulocytes generally refers to enucleated erythroid cells that are not yet fully biconcave, they represent a heterogeneous population of all stages in between enucleated reticulocytes and erythrocytes.
• Reticulocytes may be divided into 4 groups according to their relative proportion of mRNA (maturation) as judged by a progressive reduction in the compactness of the reticulum (the fourth group being the most mature). At steady state erythropoiesis the relative frequency of these groups are:
  • Group I <1%
  • Group II <1%
  • Group III 30%
  • Group IV > 60%
• The reticulocyte count in the peripheral blood is a result of three variables:
  • The rate of release of reticulocytes from the bone marrow.
  • The degree of immaturity of the freshly released reticulocytes.
  • The rate of disappearance of reticulum. 

Methods of reticulocyte enumeration

• Examination of a standard Wright-Giemsa:
  • In the standard peripheral smear, reticulocytes are seen as polychromatic cells (they are not called reticulocytes because they do not show presence of reticulum in the cytoplasm). 
  • According to the College of American Pathologists, polychromatophilic cells are:
    • Nonnucleated, round, or ovoid red cells staining homogeneously pink-gray or pale purple (owing to residual RNA in the cytoplasm).
    • Larger than mature RBCs and usually lack central pallor.
  • Detects only the most immature of the reticulocytes (i.e., Group I or II cells, namely those with the most RNA).
  • The method is not sensitive enough to be used for reticulocyte quantitation.

 

• Manual method for counting reticulocytes:
  • From the late 1940s until the early 1980s, reticulocyte enumeration was performed solely by microscopic examination of supravitally stained peripheral smears. 
  • In the United States the most popular supravital staining technique for reticulocytes is the new methylene blue method, first described in the late 1940s:
    • A few drops of a 1.0% (w/v) saline (NaCl, 0.152 mol/l) solution of New Methylene Blue (NMB) or Brilliant Cresyl Blue are mixed with an equal volume of peripheral blood collected in EDTA (1.5 mg/dl).
    • The solution is mixed and using a pipette or other transfer device an evenly thin film of stained blood is made on a 2.5 x 7.5 cm microscope slide.
    • Staining is rapid (1 or 2 minutes).
    • After the film has dried, an area of the film where the cells are undistorted and the reticulocytes appear to be well-stained is chosen for examination. 
    • 100-1000 erythrocytes are examined and the proportion of erythrocytes containing reticulin is determined (may take the form of a delicate network, a collection of fragments and rods, a few scattered dots, or diffuse fine granules).
    • Reticulocytes are defined as any nonnucleated red cell which contains two or more particles of blue-stained material, visible without fine microscope adjustment, after exposure to supravital stains, such as brilliant cresyl blue or new methylene blue.
  • Reticulocyte count is reported as % of total red blood cells.
  • Disadvantages:
    • Tedious
    • Time consuming
    • Subjective with high inter-observer variation and poor coefficients of variation.
    • High sampling error owing to the limited number of cells that can be counted:
      • When the reticulocytes are only 1% of the population and only 100 red cells are counted in total then it can be predicted that the observed proportion may be anywhere between 0% and 3%.
      • When the reticulocytes are 10% of the red cell population but only 100 red cells are counted then the observed result may lie between 3% and 16%.
    • Distinguishing the most mature and least reticulated reticulocyte from a mature red blood cell can be difficult.
    • Staining anomalies
    • Pappenheimer bodies, Heinz bodies may be mistaken for reticulocytes.

• Automated reticulocyte analysis: 
  • Development of RNA-specific fluorescent dyes in the 1980s provided a means by which to use the flow cytometer for reticulocyte enumeration.
    • The methodology for counting reticulocytes is based on the application of
      automated fluorescent flow cytometry, utilizing, for instance, thiazole orange or polymethine dye which
      binds to cytoplasmic RNA in the reticulocytes.
    • The flow cytometric quantitation of reticulocytes depends on the measurement and discrimination of cell size by forward light scatter and RNA content by a specific RNA fluorochrome.
  • Advantages:
    • The identification is based directly on the presence of RNA within the cells rather than on some aspect of polysomal remnants.
    • Rapid analysis of thousands cells in seconds.
    • Greater analytic precision.
    • Greater precision, accuracy, and reproducibility.
    • Less costly than manual reticulocyte counting.
  • Flow cytometric analysis was also found to provide clinically valuable information not available by light microscopy:
    • Because the measured fluorescence intensity is directly proportional to the amount of RNA in the immature erythrocytes, this method has the ability to quantitate reticulocyte maturity:
      • Quantitation of RNA in the reticulocytes using fluorescence intensity of thiazole orange-stained whole blood samples, termed the reticulocyte maturity index (RMI).
      • In general, those patients with a greater degree of anemia or significant reticulocytosis were found to have an increased RMI, indicative of the physiologically expected response of increased erythropoiesis and the release of newly produced and “stress” reticulocytes into the peripheral circulation.
    • Special reticulocyte parameters
      • Mean cellular volume of reticulocytes (MCVr)
      • Mean cellular Hb content of reticulocytes (MCHr)
      • Mean cellular Hb concentration of reticulocytes (MCHCr)
  • Because the cells are recognized on a different basis from that by which reticulocytes are primarily defined, the two features may not always identify the same cells It may now be more appropriate to regard ‘reticulocyte’ as a synonym for immature red cells which have a number of characteristics: namely, they contain residual RNA, have residual transferrin receptors on their surface, and may develop a reticulum when exposed to certain supravital stains.

Reporting reticulocyte counts

• The reticulocyte count may be expressed as a percentage or in absolute terms (number of reticulocytes in unit blood volume).
• In order to derive a more meaningful expression of effective marrow production, formulas have been proposed to correct the crude reticulocyte percentage for:
  • Degree of anemia
  • Maturation time

Crude reticulocyte percentage

• Reticulocyte count may be reported as a % of the total red cell count as determined by manual counting or automated flow cytometry.
• The normal mean percentage reticulocyte count by light microscopy is 1.0% to 1.5%, with 3% being the upper limit of normal. 
• The crude reticulocyte percentage is dependent not only on the number of reticulocytes in circulation but also on the number of red cells (% retics = retic count/RBC count) and on the reticulocyte maturation time.

Reticulocyte index (corrected reticulocyte count [CRC] or packed cell volume correction)

• The percentage reticulocyte count can increase either because there are more reticulocytes in the circulation, or because there are fewer mature red blood cells circulating, as in anemia. 
• For this reason, the observed reticulocyte count is corrected to a normal Hct, Hb or red blood cell count.
• For example, if the crude reticulocyte count is 2% in a patient with a Hct of 22.5, the corrected count or reticulocyte index is 2% x 22.5/45 = 1% (assuming a normal Hct of 45%).

Reticulocyte production index (RPI) (reticulocyte maturation index, shift correction)

• In patients with anemia, reticulocytes are released earlier from the bone marrow into the circulation. Upon being released prematurely, reticulocytes must complete the maturation processes in the bloodstream. So, instead of spending 3 days in the bone marrow and one day in the peripheral blood (before maturing into a red blood cell), they might spend only 1 or 2 days in the bone marrow and 2-3 days in the circulation. 
• Early-release reticulocytes are called shift reticulocytes or stress reticulocytes. This immaturity is recognizable on the blood smear through the relative increase in size and basophilia of the ‘shift’ cells.
• Any increase in the reticulocyte maturation time within the circulating blood will result in an overestimate of the reticulocyte count due to the accumulation of such cells in the blood. Stated another way, the number of reticulocytes may be markedly increased in the blood without any increase in the erythropoietic activity in the marrow.
• Some, but not all, studies have shown an inverse correlation between the the degree of immaturity of circulating reticulocytes and the degree of anemia. 
• In 1993, Ivor Cavill wrote: “Hillman & Finch (1969) sought to express the interaction between the rate of reticulocyte production, maturation and hematocrit as a Reticulocyte Production Index which required an estimate of the maturation time of the reticulocyte. However, the calculation of reticulocyte maturation times in vivo, in an admittedly small number of patients, showed that these ranged between 0.5 and 2.5 d [referring to a previous study of the author’s] and that there was no relationship between the maturation time and the hematocrit“.
• There are certain pathological states, e.g., renal failure, where the erythropoietin output is not consistent with the degree of anemia present, and the expected prolongation of reticulocyte maturation time does not occur. For the shift correction to be valid, there must be a normal relationship between the degree of anaemia and the increased erythropoietin concentration which produces the shift. This is validated by finding a high percentage of basophilic macroreticulocytes, or ‘shift’ cells, in the peripheral blood film The RPI is an effort to correct for reticulocyte maturation time.
• An approximate correction for shift visible on the blood smear is to assume that the reticulocyte lifespan has been doubled and therefore to divide by 2. 
• The normal range for the reticulocyte production index is the same as for the uncorrected reticulocyte count since the normal maturation time in the peripheral blood is 1 day.
• An RPI of more than 3 indicates an appropriate bone marrow response (regenerative anemia), while an RPI of less than 2 indicates an inadequate compensatory response to correct the anemia (arregenerative anemia). Elevated reticulocyte production indices ( > 3) are associated with chronic hemolysis, recent hemorrhage, or response to therapy. Depressed indices (< 2) are generally associated with either marrow failure or ineffective erythropoiesis, as in the megaloblastic anemias due to vitamin BI2 or folate deficiencies.
• RMI may be estimated not only by manual counting of polychromatophilic cells on a peripheral smear but also by automated quantitation of reticulocyte staining intensity. Implementation of flow cytometry methods and use of Thiazole Orange (TO) to visualize RNA provided a more advanced reticulocyte maturity index classification system.
• When the two corrections are applied together, the reticucyte production index is obtained

Forget the percentage and correction factors and focus on the absolute reticulocyte count:

Absolute reticulocyte count

• A far easier and more direct way to correct for the Hct/Hb/RBC count is to consider the absolute reticulocyte count, which is provided by automated hematology counters. 
• The absolute reticulocyte count autocorrects for the degree of anemia. It does not correct for maturation time. However, as discussed above, and as elaborated on in Knowledge Check, the value of the correction factor for maturation time is suspect.
• The absolute reticulocyte count is normally between 25,000 and 100,000 reticulocytes/mL
• Reticulocyte counts as high as 350,000/microL (3.5 x 10¹¹/L) are possible.
• Most importantly an absolute reticulocyte count > 120 x 10⁹/L is considered an appropriate response to anemia, while an absolute reticulocyte count < 120 x 10⁹/L is considered an inappropriate response to anemia.

Threshold values for various reticulocyte counting parameters

Parameter	Formula	Threshold
Crude reticulocyte %	N/A	Normal range of 0.5 to 2 percent in the absence of anemia. Not sufficiently discriminatory in cases of anemia.
Reticulocyte index	Reticulocytes (%) × (observed patient HCT [percent] ÷ 45 [percent])	< 2, underproduction; > 3 appropriate marrow response
Reticulocyte production or maturation index	Corrected reticulocyte count (%) ÷ maturation correction factor* (days). * Maturation correction (in days): HCT >35%: 1; HCT 26 to 35%: 1.5; HCT 16 to 25%: 2; HCT <15%: 2.5.	< 2, underproduction; > 3 appropriate marrow response
Absolute reticulocyte count	Reticulocytes (%) × RBC count (millions of cells/microL)	< 120 x 109/L, underproduction; > 120 x 109/L, appropriate marrow response

Some historical miletsones

Year	Discovery	Reference
1865	Wilhelm Heinrich Erb (1840–1921), a German neurologist, noted granules in red blood cells of humans and animals made anemic by venesection. He used picric or acetic acid to demonstrate these granules. Erb, however, erroneously regarded these cells as transitional forms between white and red corpuscles.	Erb, W. Virchows Arch Pathol. 1865;34:138-193
1881	Paul Ehrlich (1854–1915) was probably the first investigator to describe the cells now regarded as reticulocytes. He showed granular basophilic filamentous substance in the red corpuscles in normal blood and in anemia using intravital staining with methylene blue. He described the stained material as fine, dense, and elegant networks. He considered the basophilic substance as a feature of senescent erythrocytes rather than of young ones.	Erlich P: Blut. Berl Klin Wschr. 1881;18;43
1893	Askanazy noted a reticulated substance in the erythrocytes of a rapidly progressive anemia	Askanazy, S.: Ztschr. f. klin. Med. 1893;23:80
1922	In 1922, Edward Bell Krumbhaar (1883–1966), the eminent leader of several American medical societies, first coined the term “reticulocyte” when he stated that: “Erythrocytes revealing a more or less extensive reticulum (granular filamentous substance) by the methods of vital staining may be conveniently designated ‘reticulocytes’.	Krumbhaar EB, Chanutin A. Experimental Biology and Medicine. 1922;19:188-190
1932	Ludwig Heilmeyer (1899–1969), a German internist, published the classic descriptions of reticulocyte morphology at different stages of maturation, indicating phenotypic diversity within the reticulocyte population. He and Westhauser divided the cells into 4 groups (the fourth being the most mature), designated by Roman numerals and characterized by a progressive reduction in the compactness of the reticulum.	Heilmeyer L and Westhauser R. Ztsch. Kline. Med. 1932;121:361
1949	The supravital staining technique described by Brecher remains the standard method for reticulocyte enumeration	Brecher G. Am. J. Clin. Pathol. 1949;19: 895
1969	Clinical importance of correcting for the presence of stress reticulocytes developed by Hillman	Hillman RS, J. Clin. Invest. 1969;48:443-53
1989	The automation of reticulocyte counting started in 1989, when Toa Medical introduced a benchtop Sysmex R-1000 reticulocyte analyser in particular for counting reticulocytes.	N/A