Reticulocyte stains are used to identify immature red blood cells (reticulocytes) by precipitating their residual RNA. A stain is prepared using new methylene blue and potassium oxalate or commercial stains can be purchased. Equal volumes of blood and stain are mixed and incubated before making blood films. Reticulocytes are identified and counted under a microscope by their blue aggregates or granules of precipitated RNA. Counts of reticulocytes are an indicator of bone marrow response to anemia. The percentage of reticulocytes correlates with the level of polychromatophilic erythrocytes seen on blood films.

1. Reticulocyte Stains
CHAPTER 1 I EXAMINATION OF BLOOD SAMPLES 13
Reticulocyte stains are commercially available. Those wishing to prepare their
own stain can do so by dissolving 0.5 g of new methylene blue and 1.6 g of
potassium oxalate in 100 mL of distilled water. Following filtrations, equal
volumes of blood and stain are mixed together in a test tube and incubated at
room temperature for 10 to 20 minutes. After incubation, blood films are made
and reticulocyte counts are performed by examining 1,000 erythrocytes and
determining the percentage that are reticulocytes." The use of a Miller's disc in
one of the microscope oculars saves time in performing the reticulocyte count.
The blue-staining aggregates or "reticulum" seen in reticulocytes (Fig. 4D)
does not occur as such in living cells but results from the precipitation of
ribosomal ribonucleic acid (RNA; the same RNA that causes the bluish color
seen in polychromatophilic erythrocytes) in immature erythrocytes during the
staining process.I I As a reticulocyte matures, the number of ribosomes decreases
until only small punctate (dotlike) inclusions are observed in erythrocytes
(punctate reticulocytes) stained with the reticulocyte stain (Fig. 4E). To reduce
the chance that a staining artifact would result in misclassifying a mature
erythrocyte as a punctate reticulocyte using a reticulocyte stain. the cell in
question should have two or more discrete blue granules that are visible without
requiring fine-focus adjustment of the cell being evaluated to be classified
as a punctate reticulocyte.
In normal cats. as well as in cats with regenerative anemia. the number of
punctate reticulocytes is much greater than that seen in other species." This
apparently occurs because the maturation (loss of ribosomes) of reticulocytes in
cats is slower than that in other species. Consequently, reticulocytes in cats are
classified as aggregate (if coarse clumping is observed) or punctate (if small
individual inclusions are present). Percentages of both types should be reported.
Based on composite results from several authors, normal cats generally have
from 0% to 0.5% aggregate and 1% to 10% punctate reticulocytes when determined
by manual means. Higher punctate numbers of 2% to 17% have been
reported using flow cytometry."
The percentages of aggregate reticulocytes in cats correlate directly with
the percentages of polychromatophilic erythrocytes observed in blood filmsIn contrast to those of
the cat, most reticulocytes in other species are of
the aggregate type. Consequently, no attempts are made to differentiate stages
of reticulocytes in species other than the cat. The percentage of reticulocytes in
most species correlates directly with the percentage of polychromatophilic erythrocytes
observed on routinely stained blood films.
Heinz bodies are composed of denatured, precipitated hemoglobin. They
are spherical, stain pale blue with reticulocyte stains, and are usually found at
the periphery of the erythrocyte.
New Methylene Blue "Wet Mounts"
A new methylene blue "wet mount" preparation can be used for rapid information
concerning the number of reticulocytes, platelets, and Heinz bodies
present. The stain consists of 0.5% new methylene blue dissolved in 0.85%
NaCl. One mL of formalin is added per 100 mL of stain as a preservative. This
stain is filtered after preparation and stored in dropper bottles. Alternately, the
stain may be stored in a plastic syringe with a 0.2 IJ-m syringe filter attached so
that the stain is filtered as it is used. Dry unfixed blood films are stained by
placing a drop of stain between the coverslip and a glass slide. This preparation
is not permanent and does not stain mature erythrocytes or eosinophil granules.
Punctate reticulocytes are not demonstrated, but aggregate reticulocytes appear
as erythrocyte ghosts containing blue to purple granular material (Fig. 4F).
Platelets stain blue to purple, and Heinz bodies appear as refractile inclusions
within erythrocyte ghosts. Although this staining method is not optimal for
differential leukocyte counts, the number and type of leukocytes present can be

2. appreciated.
MANUAL RETICULOCYTE COUNT PROCEDURE
8
A.
PRINCIPLE:
The reticulocyte is a non-nucleated immature red cell containing residual RNA. A
supravital stain,
new methylene blue
, is used to precipitate the RNA into dark-blue f ilaments
or granules to identify retics.
B.
SPECIMEN and REAGENTS
: EDTA w hole blood is the preferred anticoagulant; New
Methylene Blue Staining solution, 12x
75mm tubes, pipets, glass slides.
C.
PROCEDURE:
1. Put 2 drops of new methylene blue in the bottom of a 12x75mm tube. Using a pipette,
add 2 drops of w ell-mixed EDTA blood to the tube.
2. Mix blood/stain mixture. The mixture colo
r should be smoky-gray. Adjust if needed,
i.e., add more blood if mixture is too blue.
3. Incubate mixture at least 5 minutes but no longer than 10 minutes.
4.
MIX
solution again....important! Prepare 2-4 good smears,
LABEL
and let dry.
5.
Counting:
Using
oil/100x
pow er, count 500 total red cells separating mature RBC's f rom
retics (use tw o counter keys). Retics are greenish with blue precipitates of RNA.
Tw o
“dots” or more is a retic.
Go f rom feather edge to body of smear, making sure you are
not counting too thick.
6. Tw o techs count 500 RBC's on dif ferent retic
smears for a total of 1000 RBC's counted.
7.
Quality control:
The number of retics/500 RBC's must agree +
2 retics betw een techs
to accept results or another slide is count
ed. Controls must read w ithin the assayed
range to accept results.
8. Both a relative percent retic and an absolute retic are reported:
a. Relative
number
- # of retics in total of 1000 RBC's =
percent (%)
.
b. Absolute
number
- retic % x the RBC count/cmm =
thousands/cmm
.
D.
CALCULATIONS:
1. The relative reticulocyte count uses the su
m of the tw o techs answer
s and the percent is
reported to the nearest tenth (one decimal):
# retics in 1000 RBCs
=
% OR
# retics

3. x 100 =
%.
10 1000 RBCs
2. The absolute reticulocyte count is repo
rted to the nearest thousand/cmm using the
follow ing calculation:
# retics/cmm = # retics
x RBC millions/cmm
OR
retic %
x RBC/cmm
1000 RBCs 100
E.
SOURCES OF ERROR:
1. Inadequate mixing before making smears
2. Counting artifact or other inclusions as retics......black/shiny inclusions are “junk”.
3. Improper ratio of blood to stain.
4. Not counting all of the retics.....two
blue “dots” or more is a retic.
5. Wrong calculati
EXAMINATION OF STAINED BLOOD FILMS
An overview and organized method of blood film examination are presented
here. Descriptions and photographs of normal and abnormal blood cell morphology,
inclusions, and infectious agents will be given in subsequent sections.
Blood films are generally examined following staining with Romanowskytype
stains such as Wright or Wright-Giemsa stains. These stains allow for
examination of erythrocyte, leukocyte, and platelet morphology. Blood films
should first be scanned using a low-power objective to estimate the total
leukocyte count and to look for the presence of erythrocyte agglutination (Fig.
SA), leukocyte aggregates (Fig. SB), platelet aggregates (Fig. SC), microfilaria
(Fig. SD), and abnormal cells that might be missed during the differential
leukocyte count. It is particularly important that the feathered end of blood
films made on glass slides be examined because leukocytes (Fig. SE) and platelet
aggregates (Fig. SF) may be concentrated in this area. Aggregates of cells tend
to be in the center of coverslip blood films rather than at the feathered edge.
When examining a glass-slide blood film, the blood film will be too thick
to evaluate blood cell morphology at the back of the slide (Fig. 6A) and too
thin at the feathered edge where cells are flattened (Fig. 6C). The optimal area
for evaluation is generally in the front half of the smear behind the feathered
edge (Fig. 6B). This area should appear as a well-stained monolayer (a field in
which erythrocytes are dose together with approximately one half touching each
other) of cells.
Hemoglobinometry Hemoglobin Determination Decrease in
hemoglobin concentration beyond established normal ranges for
age and sex is called “ anemia”, whereas increase in hemoglobin
concentration beyond established normal ranges for age , sex, and
geographical distribution is called “polycythemia”. So that, for
correct diagnosis it is important to determine accurately and
precisely hemoglobin concentration. Many methods are available
for the determination of hemoglobin, but among them the relevant,
and the recommended one is the Modified Drabkin’s Method.

4. ICSH (International Committee for Standardization in Hematology)
consider this method as the reference method for hemoglobin
determination. Drabkin’s solution contains the following:-
1- Potassium Ferricyanide
2- Potassium Cyanide.
3- Non- ionic Detergent
4- Dihydrogen Potassium Phosphate.
Well mixed EDTA anticoagulated blood is diluted in Drabkin’s
solution; non-ionic detergent will lyse the red cells to (1) liberate
hemoglobin, and to (2) decrease the turbidity caused by red cell
membrane fragments by dissolving them. Then, hemoglobin is
oxidized and converted to methemoglobin (Hi) by potassium
ferricyanide, this step is accelerated by the dihydrogen potassium
phosphate, and requires approximately 3 minutes for total
conversion. Potassium cyanide will provide cyanide ions to form
cyanomethemoglobin (HiCN), which have a broad spectrum of
absorption at 540 nm. The absorption can then be compared with
a hemoglobin standard with a known hemoglobin
concentration, and by applying Beer’s law extract the hemoglobin
concentration of the unknown (i.e. the patient).
Hemoglobin + Potassium Ferricyanide Methemoglobin (Hi)
Methemoglobin + Potassium Cyanide Cyanomethemoglobin