Principle
Reticulocytes are juvenile red cells; they contain remnants of the ribosomal ribonucleic acid (rRNA). Ribosomes have the property of reacting with certain basic dyes, such as azure B, brilliant cresyl blue or new methylene blue to form a blue or purple precipitate of granules or filaments. This reaction takes place only in vitally stained, unfixed preparations (metachromatic stain).
Staining solution
1) Dissolve 1 gram of new methylene blue or azure B in 100 ml of iso-osmotic phosphate buffer pH 6.5.
2) Filter the solution and store it at 4 degrees celsius.
Method
1) Deliver 2 or 3 drops of the dye solution into a 75 x 10 mm plastic tube by means of a plastic Pasteur pipette.
2) Add 2-4 volumes of patient’s anticoagulated blood to the dye solution and mix. There is a greater amount of blood to be taken in anemic patients and a lesser amount in polycythemic patients.
3) Keep the mixture at 37 degrees celsius for 15-20 minutes.
4) Resuspend the red cells by gentle mixing and make film on glass slides in the usual way
with inberte
5) Allow to dry and examine without counterstaining.
Counting
Choose an area of the film where the cells are undistorted and the staining is good. Using 100x oil immersion lens, count the number of reticulocytes seen per 1000 red cells.
Counting of red cells can be helped by inserting into the eyepiece a paper or cardboard diaphragm, in the centre of which has been cut a small square with sides about 4 mm in length. An easier time-saving method is to use a Miller-Ocular insert. This is an eyepiece giving a square field, in the corner of which is a smaller ruled square, one ninth the area of the total square. Reticulocytes are counted in the large square, and the total numbers of red cells is counted in the small square. Reticulocyte counts should be done in large squares, and RBC counts should be done in small squares.
Calculation
Number of Reticulocytes in N field = X
Average number of red cells per field = Y
Total number of red cells in N fields = N x Y
Reticulocyte % = [X/(N x Y)] x 100%
Absolute Reticulocyte count = % x RBC
Result
Reticulocytes are identified as non-nucleated red cells that contain at least 2 blue-staining particles or one particle linked to a filamentous thread. Reticulocytes can be classified into four groups, ranging from the most immature reticulocytes with a large clump of reticulin (group 1), to the most mature, with a few granules of reticulin (group IV).
Normal range
In adults and children: 0.5–2.5%
In infants: 2–5%