In light microscopy, the sample must first be supravitally stained, usually using new methylene blue or brilliant cresyl blue, which binds to the RNA. The stain allows cells to be recognized by the blue intracytoplasmic precipitate, ranging from the appearance of granules to a network of reticular material.[2] It is mixed with equal parts whole blood and incubated at room temperature. Slides preparation is with a Wright counterstain, which helps illuminate the distinction of the reticulocytes with the background. The tester manually counts the reticulocytes under the electron microscope.[6][1]

Measuring reticulocyte count with light microscopy is not commonly used anymore. It has low reproducibility and can be unreliable with a coefficient of variation ranging from 25% to 48%.[7]

Electron microscopy gives a more detailed view of individual reticulocytes and allows for the organizational changes to be followed during their life span. Cell preparation is by fixing in a glutaraldehyde/tannic acid solution in sodium cacodylate followed by resuspension in 2.0% agar. Agar slices are appropriately washed and then stained with 1.5% uranyl acetate in 50% methanol and lead citrate.[8]

In the beginning phase of reticulocytes, it is possible to visualize mitochondria (including heme synthesis), many ribosomes, endocytic vesicles, intracellular vesicles, and clathrin-coated pits. Microscopy also shows that there is a much higher rate of iron incorporation in the early stages of reticulocytes. In more mature reticulocytes, one can see the degradation of the mitochondria, compaction of hemoglobin, and the presence of more intracellular vacuoles containing debris and the exocytic vesicles.[8]

Reticulocytes are as a useful clinical indicator of anemias and bone marrow response to anemia. Reticulocyte count in a healthy person should be between 0.5 to 2.5%.[9] When a patient is anemic, and the bone marrow is unable to respond, reticulocyte count will be low. When the bone marrow can respond appropriately, the reticulocyte count will increase.[1]

A decrease in reticulocyte count in patients can be caused by[1][3][1]:

• Hypochromic anemias: Iron deficiency anemia, sideroblastic anemia, thalassemia, and anemia of chronic disease are all causes of a decreased reticulocyte count because they result in decreased hemoglobin synthesis.
• Aplastic anemias: This is pancytopenia in response to bone marrow failure. While there are many causes of this, they all diminish erythropoiesis as reticulocytes form in the bone marrow. [1]
• Nutritional deficiency: Insufficient amounts of vitamin B12 and folate are causes of megaloblastic anemia. These nutrients are needed in the bone marrow to synthesize DNA and without will cause a decrease in reticulocyte production.
• Aplastic crisis in hemolytic anemia: Aplastic crisis is a transient reduction in erythropoiesis due to a decline of precursors in the bone marrow. This is usually the result of an infection such as parvovirus B19, salmonella, and Streptococcus pneumoniae. Patients with hemolytic anemias have elevated reticulocyte counts, so when they present with a low count, it is possibly an aplastic crisis.
• Myelodysplastic syndromes: These syndromes characteristically demonstrate bone marrow damage from multiple causes. The erythropoietic cell line is usually affected, and the bone marrow is unable to produce reticulocytes.

Causes of increased reticulocyte count include[1]:

• Hemolytic anemias: These anemias are a result of red blood cell destruction. The bone marrow is still functional and has the required components to create reticulocytes. It responds to the anemia by increasing the synthesis of reticulocytes.
• Blood loss: An acute or chronic blood loss will cause an increase in erythropoiesis.

Reticulocyte analysis, mainly immature reticulocyte fraction (IRF), has also been used in chemotherapy patients with leukemia to determine the regenerative activity of the bone marrow during and after treatment. This same idea applies to bone marrow transplant patients.[9]

Another clinical use of reticulocyte count is in patients taking hydroxyurea for sickle cell anemia. This medication impairs bone marrow production of reticulocytes, therefore, causing a decrease in the count. For this reason, frequent reticulocyte counts are necessary while on this medication.[10]

In end-stage renal disease patients receiving EPO therapy, the reticulocyte count has been used as a parameter to measure the response to therapy.[1]

Other values apart from reticulocyte count that can be calculated to assess function:

• Reticulocyte production index (RPI) = [%reticulocyte count x Patient Hct]/45(normal Hct)
  • This is a corrected reticulocyte count used in anemias. In anemias, a falsely elevated reticulocyte count can occur. A decrease in RBCs automatically increases the reticulocyte count even if erythropoiesis has not taken place because the count represents a percentage in comparison to RBCs. It is possible the appropriate increase of erythropoiesis has not taken place in response to the anemia. This value, therefore, corrects for this issue.[2]
• Immature reticulocyte fraction (IRF): This is a value of the immature reticulocytes measured as a ratio of immature reticulocytes to the total amount of reticulocytes.[11]
• Reticulocyte hemoglobin content (Chr): This is a useful indirect measure of iron deficiency anemia. It measures the amount of the iron available in the bone marrow that can be used by the reticulocyte for the production of hemoglobin. Multiple studies have shown a Chr of less than 28 pg is a good predictor for iron deficiency anemia.[12]