Reticulocytes are immature red blood cells (RBCs) produced in the bone marrow and released into the peripheral blood where they mature into RBCs within 1 to 2 days. An increase or decrease in reticulocyte count can be an indicator of erythropoiesis activity or failure of, especially relative to anemias and bone marrow dysfunction.
Flow cytometry is the most typical method of counting reticulocytes; this is an automated practice that provides a faster and more precise way to measure reticulocyte count. The first step in the proces is reticulocytes staining by mixing thiazole orange solution with whole blood. This solution is kept in the dark, incubated at room temperature, and then run through the laser-based machine. The stain adheres to ribosomal RNA (rRNA) which allows for differentiation of reticulocyte staging. New cells have more RNA content compared to that of more mature reticulocytes with low RNA content. The reticulocyte count determined will be a percentage of the reticulocytes in the total amount of red blood cells in the field.
The reticulocyte undergoes multiple structural changes as it transforms into a mature RBC. The process begins within the bone marrow, where an erythroblast undergoes chromatin and nuclear condensation. This process allows enucleation to take place by interacting with macrophages, forming a reticulocyte. Breakdown and expulsion of organelles begin while in the bone marrow and continue when the reticulocyte is in the bloodstream; it includes the endoplasm reticulum, Golgi apparatus, lysosomes, mitochondria, and ribosomes via both autophagic and non-autophagic pathways. Once in the bloodstream, RNA breakdown occurs facilitated by ribonucleases. Some rRNA will remain for RBC formation. Changes in cell volume and membrane remodeling are thought to occur via exosomes. All these changes occur selectively so the necessary proteins are available during the life of a reticulocyte, but can be expelled when necessary to create a mature biconcave RBC.
In comparison to a mature RBC, reticulocytes have greater volume, higher hemoglobin content, and lower hemoglobin concentration. Reticulocytes can only synthesize hemoglobin while in the bone marrow. Thus, once they enter the peripheral blood, they have the maximum hemoglobin content they can have.
A reticulocyte functions as a step in the process of erythropoiesis. It forms from a differentiated hematopoietic stem cell. Reticulocytes form in the bone marrow where they continue to develop for 1 to 3 days. They are then released into the bloodstream with a life span of 1 to 2 days before they become mature RBCs. During this time, the reticulocyte undergoes many changes to become a mature functioning RBC. An increase in erythropoietin (EPO) levels stimulates the bone marrow to increase reticulocyte production. EPO levels normally elevate for 3 to 4 days before an increase in the reticulocyte count presents.
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. 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.
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%.
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.
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.
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%. 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.
Causes of increased reticulocyte count include:
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.
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.
In end-stage renal disease patients receiving EPO therapy, the reticulocyte count has been used as a parameter to measure the response to therapy.
Other values apart from reticulocyte count that can be calculated to assess function:
|||Riley RS,Ben-Ezra JM,Goel R,Tidwell A, Reticulocytes and reticulocyte enumeration. Journal of clinical laboratory analysis. 2001; [PubMed PMID: 11574956]|
|||Piva E,Brugnara C,Spolaore F,Plebani M, Clinical utility of reticulocyte parameters. Clinics in laboratory medicine. 2015 Mar; [PubMed PMID: 25676377]|
|||Moras M,Lefevre SD,Ostuni MA, From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals. Frontiers in physiology. 2017; [PubMed PMID: 29311991]|
|||Noulin F,Borlon C,van den Eede P,Boel L,Verfaillie CM,D'Alessandro U,Erhart A, Cryopreserved reticulocytes derived from hematopoietic stem cells can be invaded by cryopreserved Plasmodium vivax isolates. PloS one. 2012; [PubMed PMID: 22844411]|
|||Mast AE,Blinder MA,Dietzen DJ, Reticulocyte hemoglobin content. American journal of hematology. 2008 Apr; [PubMed PMID: 18027835]|
|||Riley RS,Ben-Ezra JM,Tidwell A,Romagnoli G, Reticulocyte analysis by flow cytometry and other techniques. Hematology/oncology clinics of North America. 2002 Apr; [PubMed PMID: 12094477]|
|||Preloznik-Zupan I,Cernelc P,Zontar D, Reticulocyte analysis using light microscopy and two different flow cytometric procedures. Pflugers Archiv : European journal of physiology. 2000; [PubMed PMID: 11005665]|
|||Koury MJ,Koury ST,Kopsombut P,Bondurant MC, In vitro maturation of nascent reticulocytes to erythrocytes. Blood. 2005 Mar 1; [PubMed PMID: 15528310]|
|||Raja-Sabudin RZ,Othman A,Ahmed-Mohamed KA,Ithnin A,Alauddin H,Alias H,Abdul-Latif Z,Das S,Abdul-Wahid FS,Hussin NH, Immature reticulocyte fraction is an early predictor of bone marrow recovery post chemotherapy in patients with acute leukemia. Saudi medical journal. 2014 Apr; [PubMed PMID: 24749130]|
|||Agrawal RK,Patel RK,Shah V,Nainiwal L,Trivedi B, Hydroxyurea in sickle cell disease: drug review. Indian journal of hematology [PubMed PMID: 24839362]|
|||Chang CC,Kass L, Clinical significance of immature reticulocyte fraction determined by automated reticulocyte counting. American journal of clinical pathology. 1997 Jul; [PubMed PMID: 9208980]|
|||Karagülle M,Gündüz E,Sahin Mutlu F,Olga Akay M, Clinical significance of reticulocyte hemoglobin content in the diagnosis of iron deficiency anemia. Turkish journal of haematology : official journal of Turkish Society of Haematology. 2013 Jun; [PubMed PMID: 24385778]|