RETICULOCYTE COUNT
If a patient is very anemic or polycythemic, the proportion of
Reticulocyte:
 is the last immature red blood cell stage.
 2 days in the bone marrow
 1 day in the peripheral blood before developing into a
mature red blood cells.
 contains remnant cytoplasmic ribonucleic acid (RNA) and
organelles such as the mitochondria and ribosomes.
 Is used to assess the erythropoietic activity of the bone marrow.
Principle:
 Whole blood, anticoagulated with EDTA, is stained with a
supravital stain, (new methylene blue).
 Any non-nucleated red blood cell that contains two or more
particles of blue stained granulo-filamentous material after new
methylene blue staining is defined as a reticulocyte.
Prodcedure:
1. Mix equal amounts of blood and new methylene blue stain (2 to 3
drops), and allow to incubate at room temperature for 3 to 10
minutes.
2. Remix the preparation.
3. Prepare two wedge films.
4. In an area in which cells are close together but not touching,
count 1000 RBCs under the oil immersion objective lens.
Reticulocytes are included in the total RBC count (i.e., a
reticulocyte counts as both an RBC and a reticulocyte).
5. To improve accuracy, have another laboratorian count the other
film; counts should agree within 20%.
6. Calculate the % reticulocyte count:
Or the number of reticulocytes counted can be multiplied by 0.1
(100/1000) to obtain the result.
Miller Disc
 The disc is composed of two squares, with the area of the smaller
square measuring 1/9 the area of the larger square.
 The disc is inserted into the eyepiece of the microscope and the
grid.
 RBCs are counted in smaller square
 Reticulocytes are counted in the larger square.
 A minimum of 112 cells should be counted in the small square,
because this is equivalent to 1008 red cells in the large square and
satisfies the (CAP) hematology standard for a manual reticulocyte
count based on at least 1000 red cells.
1.
dye to blood should be adjusted accordingly.
2. An error may occur if the blood and stain are not mixed
before the films are made. The specific gravity of the
reticulocytes is lower than that of mature red blood cells,
and reticulocytes settle at the top of the mixture during
incubation.
3. 3. Moisture in the air, poor drying of the slide, or both may
cause areas of the slide to appear refractile, and these areas
could be confused with reticulocytes. The RNA remnants in a
reticulocyte are not refractile.
4. Other RBC inclusions that stain supravitally including:
a) Heinz bodies are precipitated hemoglobin, usually
appear round or oval, and tend to adhere to the cell
membrane.
b) Howell-Jolly bodies are round nuclear fragments and
are usually singular.
c) Pappenheimer bodies are iron in the mitochondria
whose presence can be confirmed with an iron stain,
such as Prussian blue.
5. If a Miller disc is used, it is important to heed the “edge
rule”. A significant bias is observed if the rule is ignored.
Absolute Reticulocyte Count Principle:
 The absolute reticulocyte count (ARC) is the actual number of
reticulocytes in 1 liter (L) or 1 microliter of blood.
Calculations:
Reference Interval:
 Values between 20 x 109/L and 115 x 109/L are within the
reference interval for most populations.
Corrected Reticulocyte Count
Principle:
In specimens with a low hematocrit, the percentage of reticulocytes may be
falsely elevated because the whole blood contains fewer red blood cells.
A correction factor is used, with the average normal hematocrit considered
to be 45%.
Corrected Reticulocyte Count
Reticulocyte Production Index
Principle:
 Premature retics are called “shift reticulocytes”. These are
“shifted” from the bone marrow to the peripheral blood earlier
than usual to compensate for anemia.
 Instead of losing their reticulum in 1 day, these cells take 2 to 3
days to lose their reticula.
 When erythropoiesis is evaluated, a correction should be made
for the presence of shift reticulocytes if polychromasia is reported
in the RBC morphology.
 Most normal (nonshift) reticulocytes become mature red blood
cells within 1 day after entering the bloodstream and thus
represent 1 day’s production of red blood cells in the bone
marrow.
Reticulocyte Production Index

 The patient’s hematocrit is used to determine the appropriate

correction factor (reticulocyte maturation time in days):

Reference Interval
 An adequate bone marrow response usually is indicated by an
RPI that is greater than 3.
 An inadequate erythropoietic response is seen when the RPI is
less than 2.

Reticulocyte Control
 Several commercial controls are now available for monitoring
manual and automated reticulocyte counts.
 Most of the controls are available at three levels.

Automated Reticulocyte Counts

 Other reticulocyte parameters that are offered on some
automated instruments include:
a) Maturation index/immature reticulocyte fraction or IRF
(reflecting the proportion of the more immature
reticulocytes in the sample)
b) Reticulocyte hemoglobin concentration
c) Reticulocyte indices (such as the mean reticulocyte volume
and distribution width)
 All of the analyzers evaluate reticulocytes using optical scatter or
fluorescence after the red blood cells are treated with fluorescent
dyes or nucleic acid stains to stain residual RNA in the
reticulocytes.