Reticulocytes are immature red cells. These comprise thread like structures in the cytoplasm, which consist of Ribonucleic Acid (RNA). RNA has the property of reacting with certain dyes like brilliant cresyl blue or new methylene blue (supravital stains) to form a blue or purple precipitate of granules or filaments . New methylene blue stains the RNA filaments more intensely and uniformly and should be preferred. The number of reticulocytes in the peripheral blood represents the erythropoietic activity. Measuring reticulocyte count helps diagnose and monitor conditions such as anemia, bone marrow disorders, and response to therapy.

What Are Reticulocytes?

Reticulocytes are immature red blood cells (RBCs) that develop in the bloodstream over a period of 1-2 days. They contain ribosomal RNA, which can be stained and viewed through a microscope. An increased reticulocyte count indicates enhanced red blood cell production, whereas a decreased count suggests suppression of bone marrow activity.

Key Characteristics of Reticulocytes:

1. Size: Somewhat larger than fully matured RBCs
2. Lifespan: 1-2 days in the peripheral blood
3. Staining Characteristics: Can be stained with supravital dyes (such as new methylene blue and brilliant cresyl blue)

Why is the Reticulocyte Count Important? 

The reticulocyte count offers valuable information regarding: 

1. Bone Marrow Activity – Assesses the activity of erythropoiesis. 
2. Anemia Categorization – Aids in differentiating between hemolytic anemia (elevated reticulocyte levels) and aplastic anemia (decreased reticulocyte levels). 
3. Therapy Monitoring – Evaluates the effectiveness of treatment with iron, vitamin B12, or erythropoietin. 
4. Post-Transplant Assessment – Monitors the recovery of bone marrow following transplantation.

Methods for Determining Reticulocyte Count

1. Manual Reticulocyte Count (Microscopic Method)

• Requirements
  • Reticulocyte stain: Take 1.0 g of new methylene blue or brilliant cresyl blue and dissolve in 100 ml of citrate saline solution (0.049 g trisodium citrate dissolved in 100 ml of normal saline). Filter the mixture and it is ready for use.
  • Pasteur pipette
  • 75×10 mm plastic test tube
  • Microscope glass slide
  • Incubator or water bath at 37°C
  • Spreader
  • Microscope

Procedure

1. Mixed  2-3 drops of the patient’s EDTA anticoagulated blood with 2 or 3 drops of stain by means of Pasteur pipette into test tube.  
2. Incubate the mixture at 37°C in a water bath or in incubator for 15-20 min.
3. Re-suspend the cells by gentle mixing. Prepare smears on glass slides and air dry.
4. When films are dry, examine under a microscope using oil immersion lens.
5. Choose an area of the film where the cells are not distorted or overlapping and are properly stained. Count the reticulocytes and the RBC in the area. The field of counting can be narrowed either by using an eye piece provided with an adjustable diaphragm or inserting a piece of paper or card-board in the center of which a small square with sides about 4 mm is cut, into the eye piece. At least 100 reticulocytes are counted.

Reticulocyte % = ( Number of Reticulocytes / Total RBCs Counted  ) ×100

Example: Calculate the percentage of reticulocytes. If the number of reticulocytes seen is 100 and total red blood cells present are 2500 then reticulocyte count is equal to:

Reticulocyte % = ( 100 / 2500 ) × 100 = 4 %

Absolute reticulocyte count:

The reticulocyte count can be converted into absolute reticulocyte count, if total RBC is known, by the following formula

Reticulocytes 10⁹ /L = ( % reticulocytes × Total RBC (×10)¹² ) / 100

Corrected Reticulocyte Count:

 It is important to adjust the reticulocyte count according to the degree of anemia. This is known as the adjusted reticulocyte count.

For this purpose optimum hemoglobin (Hb%) is taken as 15 g/dl or a PCV of 0.45 L/L. Then

Corrected Reticulocyte Count % = ( Observed count ( % ) × Patient Hb (g/L)) / 150
OR
Corrected Reticulocyte Count % = ( Observed count ( % ) × Patient PCV (L/L)) / 0.45

Reticulocyte Production Index (RPI)

Adjusts for premature release of reticulocytes (shift reticulocytes) in severe anemia.

RPI = ( Corrected Retic % ) / ( Maturation Time (Days)

Maturation Time Guide:

Hematocrit (%)	Maturation Time (Days)
45	1.0
35	1.5
25	2.0
15	2.5

Interpretation:

• RPI > 2: Adequate bone marrow response
• RPI < 2: Inadequate erythropoiesis

Advantage and disadvantage of Manual Reticulocyte Counting 

What are the advantage of manual reticulocyte count ?

1. Economical 
   • Does not require costly automated equipment. 
   • Uses basic laboratory tools (microscope, staining dyes, and slides). 
2. No Advanced Machinery Requires  
   • Can be performed in settings with limited resources where automated devices are not available. 
   • Ideal for small labs operating on a tight budget. 
3. Direct Observation of Reticulocytes 
   • Permits morphological evaluation of reticulocytes and red blood cells (RBCs). 
   • Assists in identifying abnormalities (e.g., Howell-Jolly bodies, Heinz bodies) that automated devices may overlook. 
4. Adaptable and Customizable 
   • Can be adjusted with various supravital stains (e.g., new methylene blue, brilliant cresyl blue). 
   • Suitable for research and educational purposes due to its practical methodology. 
5. Calibration Not Required 
   • In contrast to automated machines, manual counting does not need regular calibration. 
   • Minimizes errors associated with equipment malfunctions. 

What are the disadvantages of manual reticulocytes count?

1. Time-Intensive 
   • Involves manual enumeration of 1,000–2,000 RBCs under a microscope. 
   • Takes between 15 and 30 minutes per sample, making it impractical for high-throughput labs. 
2. Vulnerable to Human Error 
   • Subject to differences in interpretation between observers (various technicians may count differently). 
   • Possibility of inaccurate counts due to fatigue or inconsistencies in staining. 
3. Reduced Accuracy and Consistency 
   • Not as precise as automated methods, particularly at low reticulocyte counts. 
   • Exhibits a greater coefficient of variation (CV) compared to automated systems. 
4. Limited Extra Data 
   • Does not yield immature reticulocyte fraction (IRF) or reticulocyte maturity index. 
   • Lacks additional information such as mean reticulocyte volume (MRV). 
5. Staining Discrepancies 
   • The quality of the stain impacts the visibility of reticulocytes (e.g., excessive or insufficient staining). 
   • Requires freshly collected blood samples for ideal results (delays in processing can degrade RNA). 

2. Automated Counting of Reticulocytes 

Contemporary hematology analyzers, such as those from Sysmex and Beckman Coulter, utilize flow cytometry along with fluorescent dyes for quicker and more precise outcomes.

• How It Functions: 
  • Fluorescent dyes attach to the RNA present in reticulocytes. 
  • The analyzer classifies reticulocytes according to their RNA content and size. 

• Benefits: 
  • High accuracy 
  • Reduced time for results 
  • Extra parameters available (such as the immature reticulocyte fraction – IRF) 

• Drawbacks: 
  • Costly equipment is necessary 
  • Calibration may be needed 

Normal Reticulocyte Count Ranges

• Absolute Reticulocyte Count (ARC) Adults & Children:
  • 25,000 – 75,000 reticulocytes/µL
  • 0.5% – 2.5% of total RBCs
• Newborns (Higher due to active erythropoiesis):
  • 2% – 6% (first few days)
  • Drops to adult levels by 2 weeks
• Reticulocyte Percentage (Relative Count)
  • Adults: 0.5 – 2.5%
  • Infants: (0-2 weeks) 2.0 – 6.0%
  • Children: (>2 weeks) 0.5 – 2.0%

Precautions

1. Reticulocyte count must be done on fresh blood because if blood is stored the reticulocytes will mature leading to a false low count.
2. At least 1000 red cells should be counted.
3. Reticulocytes should not be confused with HbH inclusions found in HbH disease. HbH inclusions stain paler, are dot like and occur in most of the red cells. If there is doubt, the reticulocyte count should be performed again after incubating the red cells and stain solution for another 2-4 hours. If HbH inclusions are present, the count should not decrease.
4. Heinz bodies appear as small dots present near the cell membrane and should not be confused with reticulocytes.

Clinical Interpretation of Reticulocyte Count 

Elevated Reticulocyte Count (Reticulocytosis) 

• Hemolytic Anemia (such as sickle cell disease, G6PD deficiency) 
• Acute Hemorrhage 
• Response to Therapy (iron, vitamin B12, folate treatment) 
• Post-Splenectomy 

Decreased Reticulocyte Count (Reticulocytopenia) 

• Aplastic Anemia 
• Bone Marrow Suppression (from chemotherapy or radiation) 
• Iron/Vitamin Deficiency (untreated anemia) 
• Chronic Kidney Disease (reduced erythropoietin production) 

Factors Influencing Reticulocyte Count 

• Hemolysis – Can lead to a falsely low count due to RBC destruction. 
• Splenectomy – Raises reticulocyte levels because the spleen usually removes older cells.
• Medications – Erythropoietin elevates the count; chemotherapy lowers it. 
• Sample Management – Delays in processing may impact results.