Manual Reticulocyte Procedure

Principle

The reticulocyte count is an index of bone marrow red cell production. The reticulocyte is the cell stage immediately before the mature erythrocyte. This cell spends 2 to 3 days maturing in the bone marrow before it is released into the peripheral circulation, where it spends an additional day of maturation. The reticulocyte count is the most effective measure of erythropoietic activity. Reticulocytes contain RNA and can be observed using supravital stains such as New Methylene Blue or Brilliant Cresyl Blue. Low reticulocyte counts indicate decreased erythropoietic activity. Increased reticulocyte counts indicate increased erythropoietic activity usually as the bone marrow compensates in response to anemic stress. Therefore, reticulocyte counts are a reflection of bone marrow health or injury. These counts assist physicians in diagnosis, treatment, or monitoring of patients with various anemias.

Reagents and Equipment

1. New Methylene Blue (Supravital Stain)

2. Test tubes

3. Microscope slides with a frosted end

4. Microscope with X100 (oil immersion objective)

5. Transfer pipettes

Specimen Collection and Storage

1. One EDTA tube or EDTA Microtainer

2. Specimens can be stored at room temperature for 8 hours or refrigerated at 2 to 8°C for 24 hours.

Quality Control

Commercially prepared controls are performed each day when reticulocytes are reported manually. Controls are prepared like the patient specimens and follow the procedure below for counting reticulocytes. Do not report out patient results until quality control results fall within the acceptable reference range.

Procedure

1. Mix 4 drops of New Methylene Blue with 4 drops of patient's blood. If the specimen is a small amount (such as a Microtainer), add an equal amount of stain to the Microtainer after the CBC has been completed.

2. Let the specimen mix for 10 to 15 minutes. Make a wedge smear and let it air dry. Label the smears with the patient's name, specimen number, and the date.

3. Allow the smear to completely dry and read under the microscope using X100 oil immersion.

a. Count the number of reticulocytes in 1000 cells.

b. Use the following formula to calculate the percentage of reticulocytes

Number of reticulocytes =

number of reticulocytes counted per1000cells X100 1000

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