PreLab: Reticulocyte count

• Reticulocyte


  • Stage between the orthochromatophilic normoblast (with nucleus) and a mature erythrocyte
  • Stays in the bone marrow for 2-3 days
  • Exists and matures in the circulation for 1 day before becoming a mature erythrocyte
  • First normal immature cell seen in the circulation at a very low value
  • With remnants/web/hairlike fragments; no nucleus
• Erythrocyte


  Very clear pink cytoplasm; without nucleus
• Principle of reticulocyte staining

  1. Cytoplasmic inclusions coprecipitate with the few remaining mitochondria and ferritin masses (also RNA remnants) to form visually stained dark/blue clusters with a supravital stain

  2. Mitochondria leaves RNA once precipitated

  3. Ferritin: iron stores; precipitates in the cytoplasm

  4. Cytoplasmic inclusions: RNA (mostly)/iron precipitates
• Supravital stain


  • A dye that stains living cells and its inclusions
  • New methylene blue
  • Brilliant cresyl blue
• Reticulocyte count


  • Measure of erythropoietic ability of the bone marrow
  • Indications: anemia, response to anemia, transfusion
• Sample


  • Whole blood (mixed with any anticoagulant - safer)
  • Capillary blood - performed on babies; requires smearing technique
• Supravital stains must be filtered daily or before use - may precipitate in amber bottle (confusion with remnant/contamination)
• At least 0.5-1.5% is reticulocyte in the circulation (smear)
• Reticulocyte percentage


  Increased in erythrocytosis (the reticulocyte production is increased)
• Example of reticulocyte count

  • 37 reticulocytes are found when 1000 erythrocytes are examined (37 reticulocytes, 963 erythrocytes)
• Miller ocular disc


  • A disc inserted into the eyepiece
  • Permits a less labor-intensive surveying of RBCs
  • 2 squares: A for counting reticulocytes, B for counting RBCs
  • Count at least 112 RBCs in successive fields (CAP)
• Reticulocyte count reference values

  • 0.5-2.5% (adults)
  • 4.0-6.0% (newborns)
• Clinical applications of decreased reticulocyte count

  • Aplastic anemia
  • Aplastic crisis of HA - hemolysis in the bone marrow
  • Chemotherapy and radiation and hypoproliferation
  • Pernicious anemia (Intrinsic factor absorbs vitamin B12)
  • Decreased erythropoiesis
• Clinical applications of increased reticulocyte count

  • Blood loss
  • Crisis associated with HA
  • Subsequent treatment for PA
  • Folate and iron deficiency
• Increased reticulocyte count


  Indicates hypoxia (hiking in high altitude, lack of oxygen, RBC production decreases, increased EPO)
• Reticulocyte count differentiates anemias


  • Problem in the bone marrow (defective production = anemia)
  • Increased destruction causing decreased survival (normal bone marrow but destructed in circulation)
• Polychrome-stained reticulocytes have no remnants visible
• Sources of error in reticulocyte count

  • Interobserver variation in the definition of a reticulocyte
  • Accuracy of counting of the observer (preferably, 2 laboratorians are required to count reticulocytes, results should agree within 20%, more or less)
  • Refractile bodies (due to poor drying, atmospheric moisture)
  • Precipitates (may resemble dark blue clusters in reticulocyte filter stain)
  • Inclusions (Howell-Jolly bodies: 1-2 deep purple-colored, dense structures; Pappenheimer bodies: small iron clusters)
• Causes of false decreased reticulocyte counts


  • Understaining (cause: improper incubation)
  • High glucose levels (can mask the appearance of RNA remnants)
  • Severe anemia (proportion of dye-to-blood must be adjusted accordingly)
  • Failure to re-mix after incubation, prior to smear preparation
  • Bias in the use of Miller Disc grid (use the similar inverted L rule applied in hemocytometers)
• A low reticulocyte count may indicate decreased bone marrow function due to conditions like anemia or chemotherapy treatment.
• A low reticulocyte count may indicate anemia or other conditions affecting red blood cell production, while a high reticulocyte count can be seen with hemolytic anemias or certain types of cancer.
• The reticulocyte count is an important test used to evaluate the production of new red blood cells by bone marrow, which can be affected by various factors such as iron deficiency or chronic kidney disease.