RAJESH MOHESS, CLT. A.P.

RETICULOCYTEThe 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.

Acidophilic erythroblast (NRBC) expels the nucleus and becomes the reticulocyte

Bone marrow macrophages phagocytize and digest the expelled nucleus.The size of reticulocyte is about 8 μmRNA-synthesizing ribosomes, present in reticulocytes, are stained blue

On its membrane, the reticulocyte has receptors for transferrin so that iron can enter the cell. Due to the transferrin it is also referred to as “sticky” cell

RETICULOCYTEThe reticulocyte count is the most effective measure of

erythropoietic activity.

Reticulocyte counts are a reflection of bone marrow health or injury

Reticulocytes are red cells that are non-nucleated and that contain remnant RNA material, reticulum.

Reticulum cannot be visualized by Wright’s stain; to be counted and evaluated, reticulocytes must be stained with supravital stains, like new methylene blue or brilliant cresyl blue.

RETICULOCYTEBecause the bone marrow has the capacity to expand its

production up to 7 times the normal rate, an elevated reticulocyte count or reticulocytosis is the appropriate response in anemic stress

Reticulocytes will be seen in the peripheral smear as polychromatophilic macrocytes (large, bluish cells)

Nucleated red blood cells may also be visualized in the peripheral smear as the bone marrow races to deliver cells prematurely at a rapid rate.

RETICULOCYTEReagents and Equipment

1. New Methylene Blue (Supravital Stain)2. Test tubes3. Microscope slides with a frosted end4. Microscope with x100 (oil immersion objective)5. Transfer pipettes

RETICULOCYTEProcedure

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 smear

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 (10 fields).

RETICULOCYTEUse the following formula to calculate the percentage of

reticulocytes

Number of Retic = (# retic per 1000 red cells x 100)/1000Note: 1000 red cells = 10 fieldsExample: 35 x100/1000 = 3.5%

OR Number of retic counted divided by 10Example; 35/10 = 3.5 %

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Normal Values

Adults: 0.5% to 2.0%

Infants: 2.0% to 6.0%

RETICULOCYTELimitations

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, siderocytes, and Howell-Jolly bodies can be mistaken for reticulocytes. If these are counted as reticulocytes, they will falsely increase the reticulocyte count. Inclusions should be confirmed with Wright’s stain

RETICULOCYTEInceased Reticulocyte count

Increased reticulocyte counts indicate increased erythropoietic activity usually as the bone marrow compensates in response to anemic stress

Increased count associated with:1. Rapid blood loss2. High elevation3. Hemolytic anemias4. Medications such as levodopa, malarial medications,

corticotrophin, and fever-reducing medications 5. Pregnancy

RETICULOCYTEDecreased Reticulocyte count (<0.5%)

Low reticulocyte counts indicate decreased erythropoietic activity

Decreased count associated with:1. Aplastic anemia (where the production of either

white or red cells or both is seriously impaired)2. Exposure to radiation or radiation therapy3. Chronic infection

4. Medications such as chloramphenicol, methotrexate and other chemotherapy medications5. Untreated pernicious anemia/megaloblastic anemia

RETICULOCYTECalculation of corrected reticulocyte

The reticulocyte count is most often expressed as a percentage of total red cells.

In states of anemia, the reticulocyte percentage is not a true reflection of reticulocyte production.

A correction factor must be used so as not to overestimate marrow production

Because each reticulocyte is released into whole blood containing few RBCs and a low hematocrit (Hct), the percentage of reticulocyte will be increased.

RETICULOCYTECalculation of corrected reticulocyte

The corrected reticulocyte count may be calculated by the following formula:Corrected reticulocyte count = (Retic % x patient’s Hct %) divided by Average normal HctNote: Average normal Hct is 45 for men and 42 for women

Example:

Uncorrected retic % = 5.0%Patient Hct = 35.0% (male)Average normal hct for male = 45%

Corrected retic % = (5.0% x 35%) / 45 = 3.89%Corrected retic % = 175/45 = 3.89 %

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