Reticulocytes ("retics") are young, anucleate erythrocytes, which are released from bone marrow into the blood in increased numbers as a response to anemia caused by hemolysis (destruction) or loss (hemorrhage) of erythrocytes in most species (horses are a notable exception - see more below). Identification of immature anucleate red blood cells allows us to determine if the bone marrow is responding to an anemia (given sufficient time) by increasing red blood cell production. This is termed a regenerative response. The bone marrow response is evaluated by detecting immature erythrocytes by virtue of the presence of RNA in the form of ribosomes and rough endoplasmic reticulum in their cytoplasm. The more immature the cell, the more RNA it contains. In contrast, mature red blood cells, which are no longer synthesizing hemoglobin, contain very small amounts or no RNA (they are essentially small sacs of hemoglobin).
The RNA can be detected in immature red blood cells in several ways:
Evaluation of the bone marrow response is an initial step in characterizing anemia. If the bone marrow is responding to an anemia, it is a regenerative anemia and the cause of the anemia is hemorrhage or hemolysis. If the anemia is regenerative, there will be increased numbers of reticulocytes in peripheral blood and we will observe polychromasia in the peripheral blood in all species, other than the horse. The horse releases very few reticulocytes into blood in response to a hemorrhagic or hemolytic anemia, so performing a reticulocyte count or looking for polychromasia is not helpful in this species. They do, however, release macrocytes (which may not be punctate reticulocytes) and we look for these larger red blood cells to determine if horses are responding to an anemia. Anemias without increased reticulocytes are termed non-regenerative and are due to conditions which decrease the production of erythrocytes by the marrow.
We can accurately count reticulocytes (as a percentage of total erythrocytes) using supravital or fluorescent dyes, however this is only performed routinely in the dog and cat. In the latter species, the reticulocyte percentage alone can be misleading, because a result above the reference interval may not indicate an adequate regenerative response, since the percentage alone does not take into account the severity of the anemia. To account for the degree of anemia, we usually convert the reticulocyte percentage into an absolute reticulocyte count (see below) and use this number to assess whether an anemia is adequately regenerative or not. In species other than the dog and cat, we do not routinely quantify reticulocytes but just examine a peripheral blood smear for evidence of a regenerative response, i.e. the presence of polychromatophils (or in horses, macrocytes).
With the hematology analyzer used in Clinical Pathology (ADVIA 2120),, reticulocytes are counted by staining blood a fluorescent dye (Oxazine 750). The immature red blood cells are then detected by the degree of fluorescence (mature erythrocytes will not fluoresce). We have validated this technique for the dog and cat. In addition, low numbers of reticulocytes can also be detected in the blood of horses with regenerative anemias due to experimental blood loss or after erythropoietin treatment. However, horses do not release sufficient reticulocytes for this to be clinically useful (there is a large overlap in the reticulocyte counts between non-anemic horses and horses with a regenerative anemia). In other species, reticulocytes can be counted manually using a new methylene blue stain, although this is not done routinely.
These images show canine red cell scatterplots obtained from the Advia hematology analyzer.
Right: Red cell volume by hemoglobin scatterplot. The Y-axis represents red cell volume and the X-axis represents red cell hemoglobin. Normocytic normochromic red blood cells fall in the center box (green arrowhead), whereas polychromatophilic erythrocytes (which are larger and have less hemoglobin than mature red blood cells) stream off the cluster of normal red blood cells into the upper left hand box (larger volume, lower hemoglobin, red arrow).
Left: Reticulocyte scatter plot. This represents the degree of oxazine staining (RNA) in the erythrocytes. Mature red cells (mature) take up very little oxazine. Reticulocytes have varying degrees of fluorescence, resulting in low (A), intermediate (B) and high (C) levels of fluorescence. Studies done in our laboratory have shown that the results of the combined gates (A-C) correspond well to new methylene blue-obtained reticulocyte counts in dogs and to aggregate reticulocyte counts in cats.
To determine the adequacy of the regenerative response in dogs and cats, absolute reticulocyte counts can be calculated. This is the method used at Cornell University for determining the adequacy of a regenerative response as it takes into account the severity of the anemia but does not make assumptions about a "normal" hematocrit or correction factors for reticulocyte release. The reticulocyte percentage alone is not as useful as it does not account for the severity of the anemia. The absolute reticulocyte count is calculated automatically in our laboratory from the reticulocyte % in dogs and cats (whenever a reticulocyte count is automatically added to a hemogram or a reticulocyte count is requested for a sample that also has a red blood cell [RBC] count). To calculate an absolute reticulocyte count, we use the following formula:
Abs retic count (thou/µL) = reticulocyte % (#/100) x RBC count (mill/µL) x 1000
Outlined below are guidelines for
assessing the degree of regeneration in anemic dogs and cats, along with examples of using the results. Remember that cats have 2 different types of reticulocytes, aggregate
and punctate. Only the aggregate reticulocytes are included in a reticulocyte
count (as they are the most immature and more reflective of current bone marrow
A dog with a hematocrit (HCT) of 15% and a RBC count of 1.5 mill/µL has a reticulocyte count of 3%, which is above our reference interval of 0-1.5%. Based on the reticulocyte percentage alone, you would consider the severe anemia is regenerative, however the absolute reticulocyte count is 45 thou/µL (3/100 x 1.5 x 1000), which indicates inadequate or no regeneration based on the above table. This indicates that the bone marrow is not responding appropriately to the severe anemia (as long as the bone marrow has had sufficient time to respond, i.e. 3-5 days) and that decreased production of erythrocytes is contributing to or is the main cause of the severe anemia. In contrast, a different dog with the same low HCT of 15% and RBC count has a reticulocyte count of 8%, which is also above our reference interval. The absolute reticulocyte count is 120 thou/µL (8/100 x 1.5 x 1000), which indicates a mild regenerative response and you would consider causes of blood loss or hemolysis for the severe anemia.
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