Among the great advances in cytology of the 19th century and, perhaps, the most important was Paul Ehrlich's development of polychrome staining of tissue cells. This technique provided a method to distinguish the internal structure of the blood cells and differentiate the various types of leukocytes easily. He also introduced the white cell differential count.1 This staining technique and its adaptations remain a fundamental aspect of diagnostic hematology. The prior ambiguous and inadequate state of blood cell identification was highlighted in an illustration of red and white corpuscles (plate 38, page 261) in the textbook The Microscope in Medicine printed in 1863 and authored by Lionel Beale, a British professor of medicine and pathology; the illustration was republished by Dr. Steven I. Hajdu in 2002.2
In his early studies, Ehrlich examined dried and stained blood films. He described occasional red cells that contained filamentous inclusions when stained with methylene blue. He referred to these intracellular structures as “substantia granulo-filamentosa.”3 Ehrlich thought these inclusions were cytoplasmic in nature and not nuclear remnants, and that the inclusions indicated aged red cells. Thus, Ehrlich is credited with discovering the reticulocyte; however, he thought it a marker of senescence, not a recently released cell.
In 1891, Dr. Theobold Smith and colleagues were the first to designate the reticulocyte as a “young” red cell during their studies of southern cattle fever. This disease, which at the time was of unknown etiology, devastated cattle herds in Texas and spread to exposed cattle in stockyards. Dr. Smith and colleagues discovered the causative organism in the red cell using supravital staining. The organism was later designated Babesia bigemina. These organisms were soon established to be transmitted by ticks through observation of spread among tick-infected, exposed, previously uninfected cattle. This landmark discovery introduced the concept of arthropod-borne diseases. In his studies, Dr. Smith discovered the parasites in red cells using new methylene blue stain. He also found that some red cells took up increasing amounts of stain as the cattle fever progressed and deduced they were newly formed cells, recently released from the marrow. Additionally, he established that polychromatophilic macrocytes in stained blood films represent the reticulocytes in films stained with methylene blue.4
Subsequently, it was confirmed that the reticular substance present in red cells was not residual nuclear material, distinguishing it from a type of degenerate Howell-Jolly body, a nuclear remnant. The basophilic reticulum of young red cells was also distinguished from basophilic stippling, known to be a feature of lead poisoning. In 1921, Dr. J. Albert Key reported elaborate studies showing that the reticulum in young red cells was distinguishable from mitochondria or their remnants.5 Shortly thereafter, in 1922, Dr. Edward B. Krumbhaar developed a clinically useful method of visualizing the reticulum in red cells by supravital staining with brilliant cresyl blue.6 He established a normal range of “reticulated” erythrocytes in humans and six other species. He thought the “reticulated erythrocyte” should be called “reticulocytes,” although as a matter of brevity, he thought the term “reticulosis” was preferable to “reticulocytosis.” He recognized, however, that etymological correctness favored “reticulocytosis,” and that term prevailed. In 1931, Professor Ludwig Heilmeyer defined the age of reticulocytes by the amount of their reticulum content: The denser the reticulum, the younger the erythrocyte.7 Subsequently, the reticulum in the youngest red cells was shown to be residual remnants of ribosomal RNA.
The Significance of the Reticulocyte Count and the Reticulocyte Index
In 1969, Dr. Robert S. Hillman performed quantitative studies relating the reticulocyte count to red cell production. He demonstrated that with severe anemia, the use of the reticulocyte count to determine marrow response, semiquantitatively, required a correction for premature release of reticulocytes.8 In effect, he showed that one had to convert the prevalence of reticulocytes in the blood to the incidence of reticulocytes in that 24-hour period to use the reticulocyte count as a measure of the marrow response to anemia. Under the circumstance of increased erythropoietin as in an anemia, other than that of severe chronic renal disease, marrow erythrocytes (reticulocytes) that normally mature in the marrow for approximately three days are released prematurely. Thus, reticulocytes released into the blood in the prior 24, 48, or even 72 hours, depending on the height of the plasma erythropoietin concentration and the responsivity of the marrow, are identifiable by supravital stains. It is necessary to convert the prevalence of reticulocytes to their incidence to determine the release of reticulocytes in the 24 hours under consideration, as a measure of the adequacy of the marrow response to anemia.
The maturation of the marrow erythrocyte is complex. Following enucleation, it has an approximately three-day sojourn in the marrow that is marked by degradation of its Golgi remnant, centriole, mitochondria, and most of its residual ribosomes. Cellular remodeling is extensive. Its surface area and volume are decreased substantially. There is final assembly of the membrane skeleton and loss of several cytoplasmic and membrane proteins, including tubulin, actin, surface adhesion molecules, and transferrin receptors.9 The determinants that regulate the release of four-day-old, but not one-day-old, marrow reticulocytes under steady-state conditions and the release of one- to four-day-old marrow reticulocytes under condition of elevated erythropoietin, are unknown.
Dr. Hillman's reticulocyte index, a semi-quantitative index of the adequacy of the marrow response to anemia, has been simplified with the introduction of automated counting of reticulocytes and the modern practice to report the absolute concentration of reticulocytes per 109/L in the blood, instead of reporting the percent of red cells that are reticulocytes. In the past, the percent of reticulocytes in the blood first had to be multiplied by the red cell count to determine their absolute value. In past practice before automated cell counting, because of the inaccuracies and tedium of manual red cell counting, the hematocrit was used as an analogue of red cell counts. In our era, the percent of red cells should be dispensed with in favor of the reticulocytes’ concentration in the blood. Do not use the percent of reticulocytes. Think in terms of the absolute reticulocyte count per unit volume of blood. Then, one can make the correction to convert prevalence to incidence of reticulocytes by assessing the degree of anemia. The degree of anemia is an indirect estimate of the height of the erythropoietin concentration, which governs premature release of reticulocytes. By using a factor of 1.5 to 3 based on the severity of the anemia to divide into the ratio of the absolute reticulocyte count to the normal reticulocyte count, one arrives at the reticulocyte index, a ratio that is normally approximately 1.0 and unitless.10
Applying the Reticulocyte Index
In assessing anemia, the absolute reticulocyte count and the reticulocyte index provide guidance as to the extent to which erythroid aplasia, aplastic anemia, another mechanism of suppression of erythropoiesis, or hemolysis is the principal pathophysiology at play. In the former two states of aplasia, if severe or moderately severe, the reticulocyte index is very low — between 0.0 and 0.5. It also is a critical measure of the early response to folate or cobalamin therapy where it increases significantly by day 4 to 5. In the case of therapy for iron deficiency, a significant increase occurs by day 6 to 7.
Consider two examples:
CASE 1. A 63-year-old man enters the emergency department with air hunger. He is found to have a red cell count of 1 × 1012/L (normal, 5 × 1012/L), spherocytes on his blood film, and a reticulocyte count of 500 × 109/L (normal, 50 × 109/L). A direct antiglobulin test is positive for IgG and C3. The ratio of the prevalence of reticulocyte (observed/expected) is 500/50 or 10. However, when corrected for premature release, because of the presumptive very high erythropoietin level, by dividing the prevalence ratio by three for the severity of the anemia, the resultant reticulocyte index (the incidence of reticulocytes per 24 hours) is 3.3. It has been shown by ferrokinetic studies that the acute erythropoietic response to hemolysis can increase up to approximately threefold, whereas in severe, chronic hemolytic anemias, it may increase up to sixfold.10,11 Thus, this patient's response to a profound, acute autoimmune hemolytic anemia is appropriate and robust: an approximately three-fold increase in red cell production.
CASE 2. A 48-year-old man has been followed for one year after receiving a hematopoietic stem cell transplant from a compatible donor. One year later, he began having extreme fatigue and dyspnea on slight exertion. His wife had noted pallor. When seen for these complaints, he had a red cell count of 1.2 × 1012/L, which had previously been 4.3 × 1012/L. The white cell and white cell differential count and platelet count were normal. The blood film was uninformative. The reticulocyte count was 5 × 109/L (normal, 50 × 109/L). The ratio of prevalence of reticulocytes was 5/50 or 0.1. The reticulocyte index, therefore, was lower than 0.1, a profound decrease from the normal value of 1.0. A presumptive diagnosis of erythroid aplasia was made. A marrow biopsy confirmed the marked reduction of erythroid precursors. Granulopoiesis and thrombopoiesis were normal. The hematopathologist noticed a nuclear inclusion in an infrequent large proerythroblast. A diagnosis of B19 Parvovirus infection was confirmed by polymerase chain reaction.
Of course, with a reticulocyte count of 500 × 109/L (very high) or 5 × 109/L (very low), one need not make any calculations. The diagnosis of hemolytic anemia or of erythroid aplasia is evident. However, the internist, family physician, pediatrician, gynecologist, and of course the hematologist, should be familiar with the reticulocyte index, the reasoning behind it, and should always ask in any anemic patient: “What is the reticulocyte count?” Indeed, when confronted by the presence of anemia, no matter how complex the setting, the diagnostician should initially ask these five questions:
1. What is the severity of the decrease in hemoglobin concentration? Is it mild, moderate, or profound? Making these distinctions is useful, for example, in cases of substrate deficiency states such as iron, folate, or cobalamin. The characteristic changes in red cell size and hemoglobinization are a function of the severity of the anemia. Thus, normocytic normochromic anemia at a hemoglobin level of 11 g/dL in a woman could still be iron, folate, or cobalamin deficiency as the expected red cell size and other phenotypic changes are a function of severity of the deficiency and the time of evolution of the anemia. At 6 g/dL, the characteristic phenotypic changes would be expected universally and their absence would point to a different pathogenesis.
2. What is the mean cell volume (MCV)? Is one dealing with a macrocytic, normocytic, or microcytic anemia? The consideration of this classic approach to the differential diagnosis of anemia proposed by Maxwell Wintrobe in two papers in the 1930s can be very useful, but the caveat in question #1 above must be integrated in the diagnostician's reasoning.
3. What is the reticulocyte count? Is the anemia associated with a robust erythropoietic response or is it inadequate? A distinction between hemolytic anemia and anemia of inadequate marrow erythropoiesis is an important fork in the road in one's differential diagnosis.
4. Are the white cell count and differential count and platelet count normal? That is, is the anemia isolated or is a bicytopathy (e.g., bicytopenia) or a tricytopathy (e.g., tricytopenia) present? This distinction is very important as the differential diagnosis of tricytopenia is distinctive from anemia with normal leukocyte and differential leukocyte counts and a normal platelet count.
5. What is the appearance of the red cells on the blood film? Is the chromicity (apparent hemoglobin content) or shape of the red cell abnormal? Size is best determined by the MCV since the blood film provides only two dimensions from which one can assess area instead of the three dimensions required to estimate volume. For example, distinguishing thin macrocytes from thick macrocytes and the variation in the area of the red cells on the blood film regardless of the average cell volume are complexities that are best resolved by the MCV. Examination of the blood film, however, is the singular method to identify abnormalities of red cell shape (e.g., spherocytes, elliptocytes), poikilocytes (e.g., keratocytes, dacrocytes), sickle cells, or anisochromia and anisocytosis. These features may lead directly to or contribute significantly to making a diagnosis.
The answers to these five questions should place the physician in a very strong position to pursue the specific cause of the anemia logically and, therefore, efficiently. No matter how complex the clinical situation, first answer these five questions before ordering further tests.
Recommendation
The reticulocyte count may be the only underutilized and perhaps underappreciated laboratory test. This underutilization of the reticulocyte count is especially prevalent among non-hematologists, understandably. Nevertheless, I have listened to hematologists present cases of anemia or pancytopenia and have had to ask, “What was the reticulocyte count?” The reticulocyte count should be a part of the complete blood count. As a second option, it should be reported automatically in any patient with a decreased hemoglobin concentration. Moreover, I would propose that the clinical laboratory should report the absolute reticulocyte count and the reticulocyte index with all cases of anemia. This approach would encourage physicians to apply it in their differential diagnosis and would integrate its use in the approach to the diagnosis of anemia. It would benefit patient care and the efficiency of the diagnostic approach. By adding the absolute reticulocyte count to the complete blood count, it would in fact become “complete.” In so doing, the complete blood count would contain the answer to the five key questions required in the initial approach to the diagnosis of anemia.
