Reticulocytes are the transitional cells between late juvenile erythrocyte denucleation and fully mature erythrocytes, because of its cytoplasm there is still alkaliphilic DNA material, so after live staining with brilliant tar blue staining, cytoplasm can be seen in the blue-green reticulation, so each reticulocyte RBCs.

Operation method

reticulocyte counting assay

Principle

Due to the small amount of nucleoprotein bodies and ribonucleic acid (DNA) remaining in the cytoplasm of reticulocytes, they can be stained by certain dyes (e.g., Brilliant Tar Blue, New American Blue) to form blue particles and reticulocyte material. Therefore, they can be distinguished from fully mature erythrocytes (feticulocyte diameter 8-9.5 cm).

Materials and Instruments

Blood
Neomelanin Solution
microscope

Move

First, the experimental reagents: the past with brilliant tar blue, recently more foreign use of new blue solution, because the latter reticulate material coloring is deeper, the results are more constant, brilliant tar blue staining in different specimens between the larger differences. ①, brilliant tar blue solution:

Brilliant tar blue 0.4g 38g / L sodium citrate 20.0ml sodium chloride 8.5g / L with water to 100ml ②, the new beauty blue solution:

Xinmei blue 1.0g 38g / L sodium citrate 20.0ml85g / L NaCl with water to 100ml

Second, the experimental operation:

1, in a small test tube with 2-3 drops of Xinmei blue solution or Brilliant tar blue solution and then add 2-3 drops of blood, mixed and plugged.

2, set at 37 ℃, 15-20min.

3, shake well, take the small drops pushed into the film, dry.

4、Select low magnification to choose the red blood cells scattered and evenly distributed parts, and then oil microscope counting.

5, for the convenience of counting, need to narrow the field of view, can be placed in the eyepiece of a round hard paper, the center of the opening of a small hole of about 2mm × 3mm.

6. Reticulocytes are red blood cells with reticulation, fine dots or light blue material, count the number of reticulocytes contained in 1000 red blood cells, divided by 10, that is, the reticulocytes of the hundred.

Caveat

l, reticulocytes must be in vivo, staining conditions to show, the ratio of staining and blood to 1:l is appropriate.

2， blood film preparation technology is very important, red blood cells should be evenly dispersed, such as overlap will affect the accuracy of the results. Generally, the body-tail junction of the blood film is selected for counting.Reference plant: adult: 0.008-0.02 or (25-75) × 109/L Neonate: 0.020-0.06

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