Reticulocyte count (supravital stain)

Principle

• Reticulocytes are young RBCs containing residual ribosomal RNA. Supravital dyes (new methylene blue or brilliant cresyl blue) stain living cells and precipitate the RNA as a blue network or granules.

Specimen

• EDTA blood; stable for about 24 h at room temperature or longer at 4 °C.

Procedure

• Mix equal volumes of blood and stain (e.g. 2–3 drops each) and incubate about 15–20 min at 37 °C or room temperature.
• Remix and make thin films; air dry. Do not fix; counterstaining is optional.
• Count reticulocytes among at least 1000 RBCs under oil, ideally with a Miller ocular disc.
• Any non-nucleated red cell with two or more particles of blue-stained material is counted as a reticulocyte.

Results

• Retic % = reticulocytes ÷ RBCs counted × 100.
• Absolute count = retic % × RBC count; typical adults about 0.5–2.5% or about 50–100 × 10^9/L. Local ranges apply.
• Corrected retic % = retic % × patient Hct ÷ normal Hct (0.45).
• Reticulocyte production index = corrected retic % ÷ maturation time in blood (about 1.0 day at Hct 0.45, 1.5 at 0.35, 2.0 at 0.25, 2.5 at 0.15). RPI above about 2–3 suggests an adequate marrow response.

Quality control and pitfalls

• Distinguish from Heinz bodies (pale blue, round, attached to membrane), HbH inclusions (many fine dots, golf ball appearance, need longer incubation), Pappenheimer bodies and Howell–Jolly bodies.
• Poor mixing or too few cells counted give large errors.
• Automated flow counts (RNA-binding fluorescent dyes) are more precise and give the immature reticulocyte fraction.

Clinical use

• Separates anaemia due to increased destruction or blood loss (high retic) from underproduction (low retic); monitors response to iron, B12 or folate therapy and marrow recovery.