A Miller Eye Disc is placed inside the microscope eyepiece as an aid to counting reticulocytes. This reticule is a large square inside a small square and provides the technologist the ability to isolate the reticulocytes while counting.
Reagents and Equipment
1. New Methylene Blue (Supravital Stain)
2. Test tubes
4. Microscope with Miller ocular eye disc
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.
Commercially prepared controls are performed each day when reticulocytes are reported. Controls are prepared like the patient specimen and follow the procedure below for counting reticulocytes. Do not report out patient results until quality control results are acceptable.
1. Mix 4 drops of new methylene blue with 4 drops of patient blood in a test tube. If the specimen is a small amount (such as a Micro-tainer), 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 an appropriate smear with feather edges. Label the slides with the patient's name, specimen number, and date.
3. Allow the smear to completely dry and read the slides under the microscope with oil immersion using the Miller Eye Disc (Fig. 20.5).
a. The Miller Eye Disc is a counting aid that provides a standardized area in which to count RBCs. There are two squares that make up the disc. Square 1 is nine times the area of square 2.
b. To use the disc, all reticulocytes are counted in the large (1) and the small (2) square. The RBCs are counted only in the small square.
c. Count the number of reticulocytes in 111 red cells in the small square. At this time, the counting is concluded and the number of reticulocytes is reported.
d. Use this formula for calculating reticulo-cytes in percentage. See example.
% Reticulocytes =
Total number of reticulocytes counted in large square X 100 Total RBCs in small square X 9
304 PartV • Laboratory Procedures
Conditions Associated With...
Decreased reticulocyte count
1. Aplastic anemia
2. Exposure to radiation or radiation therapy
3. Chronic infection
4. Medications such as azathioprine, chloramphenicol, dactinomycin, methotrexate, and other chemotherapy medications
5. Untreated pernicious anemia and megaloblas-tic anemia
Increased reticulocyte count
1. Rapid blood loss
2. High elevation
3. Hemolytic anemias
4. Medications such as levodopa, malarial medications, corticotrophin, and fever-reducing medications
1. Recent blood transfusion can interfere with accurate reticulocyte results.
2. Mishandling, contamination, or inadequate refrigeration of the sample can interfere and cause inaccurate test results.
3. Red cell inclusions such as Heinz bodies, side-rocytes, and Howell-Jolly bodies may be mistaken for reticulocytes. Counting these inclusions may cause a falsely elevated reticu-locyte count. Inclusions must be confirmed with Wright's stain.
See automated reticulocyte information on page
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