Histology, Reticulocytes


Article Author:
Dipti Rai
Allecia Wilson


Article Editor:
Leila Moosavi


Editors In Chief:
Kranthi Sitammagari
Mayank Singhal


Managing Editors:
Avais Raja
Orawan Chaigasame
Carrie Smith
Abdul Waheed
Khalid Alsayouri
Trevor Nezwek
Radia Jamil
Patrick Le
Anoosh Zafar Gondal
Saad Nazir
William Gossman
Hassam Zulfiqar
Hussain Sajjad
Steve Bhimji
Muhammad Hashmi
John Shell
Matthew Varacallo
Heba Mahdy
Ahmad Malik
Sarosh Vaqar
Mark Pellegrini
James Hughes
Beata Beatty
Beenish Sohail
Nazia Sadiq
Hajira Basit
Phillip Hynes


Updated:
6/14/2019 9:47:36 PM

Introduction

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.[1]

Issues of Concern

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.[2] 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.[1] The reticulocyte count determined will be a percentage of the reticulocytes in the total amount of red blood cells in the field.[2]

Structure

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.[3] Some rRNA will remain for RBC formation.[2] 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.[2][3]

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.[2]

Function

A reticulocyte functions as a step in the process of erythropoiesis. It forms from a differentiated hematopoietic stem cell.[4] 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.[5] 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.[1]

Microscopy Light

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]

Microscopy Electron

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]

Clinical Significance

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]

  • Image 11106 Not availableImage 11106 Not available
    Image courtesy Dr Chaigasame
Attributed To: Image courtesy Dr Chaigasame

Interested in Participating?

We are looking for contributors to author, edit, and peer review our vast library of review articles and multiple choice questions. In as little as 2-3 hours you can make a significant contribution to your specialty. In return for a small amount of your time, you will receive free access to all content and you will be published as an author or editor in eBooks, apps, online CME/CE activities, and an online Learning Management System for students, teachers, and program directors that allows access to review materials in over 500 specialties.

Improve Content - Become an Author or Editor

This is an academic project designed to provide inexpensive peer-reviewed Apps, eBooks, and very soon an online CME/CE system to help students identify weaknesses and improve knowledge. We would like you to consider being an author or editor. Please click here to learn more. Thank you for you for your interest, the StatPearls Publishing Editorial Team.

Histology, Reticulocytes - Questions

Take a quiz of the questions on this article.

Take Quiz
A 16-year-old male presents to the clinic with refractory hypertension and peripheral edema. His reticulocyte count is low, and Hg is 9.7g/dL. The rest of his labs are still pending. Which of the following is the site of production of signaling factor regulating bone marrow's response to anemia?



Click Your Answer Below


Would you like to access teaching points and more information on this topic?

Improve Content - Become an Author or Editor and get free access to the entire database, free eBooks, as well as free CME/CE as it becomes available. If interested, please click on "Sign Up" to register.

Purchase- Want immediate access to questions, answers, and teaching points? They can be purchased above at Apps and eBooks.


Sign Up
A 48-year-old female with menorrhagia presents to the clinic complaining of fatigue. Complete blood count reveals WBC count 5600/microL, hemoglobin 9.8 mg/dL, hematocrit 29, MCV 78.1 fl, and reticulocyte count 1.8%. Iron Studies are consistent with iron deficiency anemia. Which of the following best identifies the reticulocyte production index?



Click Your Answer Below


Would you like to access teaching points and more information on this topic?

Improve Content - Become an Author or Editor and get free access to the entire database, free eBooks, as well as free CME/CE as it becomes available. If interested, please click on "Sign Up" to register.

Purchase- Want immediate access to questions, answers, and teaching points? They can be purchased above at Apps and eBooks.


Sign Up
A slide stained with brilliant cresyl blue is being viewed by light microscopy. Many cells with blue intracytoplasmic precipitate are seen. In the cells being viewed, what is the stain adhering too?



Click Your Answer Below


Would you like to access teaching points and more information on this topic?

Improve Content - Become an Author or Editor and get free access to the entire database, free eBooks, as well as free CME/CE as it becomes available. If interested, please click on "Sign Up" to register.

Purchase- Want immediate access to questions, answers, and teaching points? They can be purchased above at Apps and eBooks.


Sign Up
A 55-year-old male with a history of alcohol use disorder presents to the clinic for a physical exam. Standard labs are drawn. His complete blood count reveals hemoglobin 10.9 g/dL and MCV 103.4 fl. He is found to have elevated homocysteine levels and normal methylmalonic acid levels. Based on this information, what would be the most likely reticulocyte count?



Click Your Answer Below


Would you like to access teaching points and more information on this topic?

Improve Content - Become an Author or Editor and get free access to the entire database, free eBooks, as well as free CME/CE as it becomes available. If interested, please click on "Sign Up" to register.

Purchase- Want immediate access to questions, answers, and teaching points? They can be purchased above at Apps and eBooks.


Sign Up
A 10-year-old female with sickle cell disease presents to the clinic for a new rash and symptoms of an upper respiratory infection. The patient denies chest pain and is afebrile. On physical exam, lungs are clear to auscultation. At the patient's last clinic visit two weeks ago, the reticulocyte count was 6.7%. Today, the reticulocyte count is 0.7%, WBC count is 9,400/microL and platelet count is 287,000/microL. What is the most likely cause of the drop in reticulocyte count?



Click Your Answer Below


Would you like to access teaching points and more information on this topic?

Improve Content - Become an Author or Editor and get free access to the entire database, free eBooks, as well as free CME/CE as it becomes available. If interested, please click on "Sign Up" to register.

Purchase- Want immediate access to questions, answers, and teaching points? They can be purchased above at Apps and eBooks.


Sign Up

Histology, Reticulocytes - References

References

Piva E,Brugnara C,Spolaore F,Plebani M, Clinical utility of reticulocyte parameters. Clinics in laboratory medicine. 2015 Mar;     [PubMed]
Mast AE,Blinder MA,Dietzen DJ, Reticulocyte hemoglobin content. American journal of hematology. 2008 Apr;     [PubMed]
Moras M,Lefevre SD,Ostuni MA, From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals. Frontiers in physiology. 2017;     [PubMed]
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]
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]
Koury MJ,Koury ST,Kopsombut P,Bondurant MC, In vitro maturation of nascent reticulocytes to erythrocytes. Blood. 2005 Mar 1;     [PubMed]
Riley RS,Ben-Ezra JM,Goel R,Tidwell A, Reticulocytes and reticulocyte enumeration. Journal of clinical laboratory analysis. 2001;     [PubMed]
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]
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]
Chang CC,Kass L, Clinical significance of immature reticulocyte fraction determined by automated reticulocyte counting. American journal of clinical pathology. 1997 Jul;     [PubMed]
Agrawal RK,Patel RK,Shah V,Nainiwal L,Trivedi B, Hydroxyurea in sickle cell disease: drug review. Indian journal of hematology     [PubMed]
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]

Disclaimer

The intent of StatPearls is to provide practice questions and explanations to assist you in identifying and resolving knowledge deficits. These questions and explanations are not intended to be a source of the knowledge base of all of medicine, nor is it intended to be a board or certification review of PA-Hospital Medicine. The authors or editors do not warrant the information is complete or accurate. The reader is encouraged to verify each answer and explanation in several references. All drug indications and dosages should be verified before administration.

StatPearls offers the most comprehensive database of free multiple-choice questions with explanations and short review chapters ever developed. This system helps physicians, medical students, dentists, nurses, pharmacists, and allied health professionals identify education deficits and learn new concepts. StatPearls is not a board or certification review system for PA-Hospital Medicine, it is a learning system that you can use to help improve your knowledge base of medicine for life-long learning. StatPearls will help you identify your weaknesses so that when you are ready to study for a board or certification exam in PA-Hospital Medicine, you will already be prepared.

Our content is updated continuously through a multi-step peer review process that will help you be prepared and review for a thorough knowledge of PA-Hospital Medicine. When it is time for the PA-Hospital Medicine board and certification exam, you will already be ready. Besides online study quizzes, we also publish our peer-reviewed content in eBooks and mobile Apps. We also offer inexpensive CME/CE, so our content can be used to attain education credits while you study PA-Hospital Medicine.