A. Purpose of examination

Reticulocytes are immature red cells normally present in small numbers in the blood (up to 2%). It increase when there is increase in erythropoietic activity. A reticulocyte count assesses bone marrow activity & value in monitoring the erythropoietic response of an anemic patient following treatment.

B. Principle of examination

The R.N.A. in the polychromatic erythrocyte is precipitated by staining them while they are still alive (without fixing the cell ). which appears as dark blue network of reticulum in some of cells (reticulocyte ).

C. Performance specifications

Not applicable.

D. Sample type required

EDTA blood.

Stored at 2℃-8℃ after analysis for 24 hrs.

E. Preservatives needed

Sample collect In EDTA bulb.

F. Reagents required:

• New Methylene Blue ——– 1.0 gm

• Sodium chloride: 0.89gm

• Distilled woter:100ml

G. Calibration method

Not applicable.

H. Detailed work bench instruction / programming steps

• Add 2 to 3 drop of RC diluting fluid into 75×10 mm plastic tubes by means of plastic Pasteur pipette. Add 2 to 3 drop of EDTA blood.

Blood: Diluting fluid ratio should be 1:1.

A larger proportion of anemic blood and smaller proportion of polycythemic blood should be added than of normal blood.

• Mix well and incubate at 37 degree celcius for 30 minutes.

• Resuspend the red cells by gentle mixing.

• Make thin film on clean glass slide and examine under oil immersion lens without fixing/counter staining.

I. Quality control procedure

• External quality assessment: Participate in external quality assessment program and maintain data in C\Records\File\2\Results of EQA and interlaboratory comparison

• Internal Quality Control: Weekly one random RC sample is checked internally by three different observer and records of comparison are kept in control C\Records\File\9\Records of internal quality control records. For any nonconformance, Procedure for identification, correction and prevention of nonconformities is activated and records are filed in C\Records\File\12\Records of Nonconformities

J. Interferences

1 Not mixing adequately the stained blood prior to making the blood film.

2 Not counting the cells accurately or counting too few cells.

3 Using precipitated stain.

K. Calculation of results and uncertainty:

Not Applicable

L. Biological reference interval

Reportable interval for examination results

M. Critical values

N. Interpretation by the laboratory

Reticulocyte count increases when there is increase in erythropoietic activity.

Reticulocyte count decreases when there is decrease in erythropoietic activity.

O. Potential sources of variability

Preanalytical sources of variability:

1. A larger proportion of anemic blood ,and a smaller proportion of polycythemic blood should be added than of normal blood.

2. Precipitated stain overlying cells may cause confusion.

3. Counts tend to decrease after 6-8hrs unless the blood is kept at 4 ℃

Reference: 1)B.J.Bain, J.Bates, M.A.Laffan; Dacie & Lewis Practical Haematlogy, twelth edition, pg27-30