Written Competency for Reticulocyte Count Answer Key
Name: ___________________
Date: _______________
Match the images of these cells stained with Brilliant Cresyl Blue with the appropriate
description.
The correct answers are on the right and highlighted.
1.
2.
3.
Hemoglobin H bodies
A.
C
Normal cell
B.
B
Reticulocyte
C.
A
Reticulocytes
RNA present in reticulocytes will appear as darker inclusions, similar to how they appear with
New Methylene Blue stain. These RNA inclusions are fewer in number than Hemoglogin H
bodies, and are only seen in reticulocytes.
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4. The arrow in this wright’s stained slide shows: Basophilic stippling, Punctuate basophilic
inclusions
Punctuate basophilic inclusions-Precipitated ribosomal RNA:Lead poisoning, Thalassemias,
Pyrimidine 5' nucleotidase deficiency.
5. The arrows in this Brilliant Cresyl Blue Stained slide show: Hemoglobin H bodies
Hemoglobin H bodies
Hemoglobin H bodies are seen as faint blue inclusions.
They appear to be on the outside of the cell, resembling sugar on a gumdrop; but they are just
inside the cell's membrane and push outward.
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6. RBCs Inclusions in this wright’s stained slide are: Howel Jolly bodies (Nuclear remenant)
7. The arrowed cell in this supravital stained slide is called: (Reticulocyte)
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8. RBCs Inclusions in this supravital stained smear are: Heinz bodies
Heinz body
 Heinz bodies are denatured globin, and represent the end-product of oxidative degradation
of haemoglobin.
 Investigation of haemolysis (when fragmented spherocytes seen on blood film) or with
history of exposure to oxidant chemical or drug.
 Fresh blood incubated with supravital stain and examined by microscopy for presence of
stained inclusions close to the red cell membrane (Heinz bodies).
 Heinz bodies may be detected post-splenectomy, with oxidative haemolysis (in normals, but
particularly in patients with G-6-PD deficiency) and in patients with unstable
haemoglobinopathies.
On supravital stained blood film which RBC inclusions are seenas?
9.
10.
11.
Description
Faint blue inclusions on the outside of the cell, resembling sugar
on a gumdrop. They are just inside the cell's membrane and push
outward.
RBC Inclusions
(d. Hemoglobin H bodies)
a. Heinz bodies
Dark blue-staining filament or at least 2 or more discrete blue
aggregates of reticulum in the erythrocyte
b. Pappenheimer bodies
(c. Reticulocytes )
Light blue-green granules and are usually present at the edge of
the erythrocyte.
c. Reticulocytes
d. Hemoglobin H bodies
(a. Heinz bodies)
12.
Several granules in a small cluster
(b. Pappenheimer bodies)
On a Wright’s- stained blood film which RBC inclusions are seen as:
13.
Description
Multiple dark blue particles or granules of varying size,
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RBC Inclusions
a. Pappenheimer
ULC/TCP/5F
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scattered throughout the cytoplasm of erythrocytes
bodies
b.Reticulocytes
c. basophilic stippling
c. basophilic
stippling
14.
Small dark purple granular bodies of varying size frequently
d.Howell-Jolly
clustered in groups of two, three or more near the edge of the
bodies
cell
(a. Pappenheimer bodies)
15.
Blue/gray RBC
(b. Reticulocytes)
16.
Single erythrocyte inclusions which are large, round, smooth
and purplish-blue staining are most likely
(d. Howell-Jolly bodies)



What is basophilic stippling?
The presence of multiple dark blue particles or granules of varying size, scattered
throughout the cytoplasm of erythrocytes in the reticulocyte stage is called basophilic
stippling. There are two types of stippling, fine or diffuse, and coarse or punctate. The
erythrocyte containing them may stain normally in other respects or it may be
polychromatophilic
What are Pappenheimer Bodies?

Pappenheimer bodies appear as small dark purple granular bodies of varying size
frequently clustered in groups of two, three or more near the edge of the cell.
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
Pappenheimer bodies, while visible on a Wright's stained smear, should be Perls' Prussian
blue stain, which is specific for iron.
What are Howell-Jolly Bodies?


Howell-Jolly bodies are round, smooth, almost pyknotic, dark purple bodies ranging
in size from 0.5 to 1.0 micron in diameter.
Located eccentrically, usually only one Howell-Jolly body occurs in a mature or
nucleated erythrocyte. Occasionally, two or more Howell-Jolly bodies per cell may
be found. These DNA inclusions demonstrate a positive Feulgen reaction which is
specific for DNA and RNA.
Characteristics of Pappenheimer bodies and Siderotic granules
What are Pappenheimer bodies?


Pappenheimer bodies are seen in the cytoplasm of mature and immature erythrocytes
on a Wright's stained smear.
They are composed of degenerating cellular remnants, which contain iron.
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



Pappenheimer bodies are most likely caused by accelerated red cell division, or
impaired hemoglobin synthesis.
 Pappenheimer bodies appear as small dark purple granular bodies of varying size
frequently clustered in groups of two, three or more near the edge of the cell.
Pappenheimer bodies, while visible on a Wright's stained smear, should be Perls' Prussian
blue stain, which is specific for iron.
Pappenheimer bodies are seen in certain types of anemia characterized by an increase in
the storage of iron, such as sideroblastic anemia and thallassemia.
These inclusions are also seen in the peripheral blood following a splenectomy. In a
healthy person with a normal spleen, Pappenheimer bodies are destroyed before the
erythrocytes enter the peripheral circulation.
Comparison of Erythrocyte Inclusions
17. In section A of the image, the arrow points to a RBC with basophilic stippling.
18. In section B, the arrows point to erythrocytes containing Pappenheimer bodies.
19. In section C, the arrows are pointing to Howell-Jolly bodies.
20. In section D, the arrow is pointing to a platelet that is sitting on top of an RBC.
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This may be mistaken for an inclusion. One of the distinguishing characteristics that can alert
you to the fact that it is not an inclusion is the halo around the platelet.
21. Arrowed RBC inclusions in this supravital stained smear are: Heinz Bodies
22. Identify RBC inclusions in this wright’s stained image.
Pappenheimer bodies
*Confirmed with Prussian stain: iron granules seen in >90% of RBCs.
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23. The large red cell in the center of this image is an example of a "shift" or stress reticulocyte.
This cell has entered the circulation earlier than normal.
Which of the following statements characterize Stress Reticulocytes?
(Choose ALL of the correct answers)
When the large reticulocytes normally found in the bone marrow are present in the
peripheral blood, they are referred to as shift or stress reticulocytes.
b. These cells may be up to twice the size of normal mature red cells and are an indication
of the bone marrow’s response to severe anemia.
c. In addition to recognizing their appearance as polychromatophlic cells on Wright’s
stained smears, it is now possible to quantify stress reticulocytes using a fluorescent stain.
d. They are classified as high, medium, or low using a fluorescent-sensitive flow cytometer.
a.
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24. Wright's stained smear. The cells that are indicated by the arrows in this are known as:
polychromatophilic red cells


The cells that are indicated by the arrows in this slide are slightly blue/gray and are
examples of polychromatophilic red cells.
Increased numbers of these cells, (approximately 2 or greater per oil immersion
field,) indicate increased red cell output by the bone marrow. Polychromatophilic
cells are larger and younger than mature red cells, and may be larger than 9 micron in
diameter. Under normal conditions, these young red cells remain in the bone marrow
one or two days before release into the bloodstream. However, when the bone
marrow is stressed due to blood loss or other conditions, these cells are prematurely
released into the blood, resulting in a blood smear with polychromasia. These red
cells are often referred to as shift cells. If stained with a supravital stain, they would
be identified as reticulocytes.
25. Arrow points to: Heinz bodies. arrowhead points to: a reticulocyte
 causes of Heinz body anemia
 http://cueflash.com/decks/bioanalytical_pathology_I
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26. In an adult male person, average reticulocyte count is 9%, hematocrit 23%, what would be
the the reticulocyte production index (RPI)? Show your calculation.
Corrected reticulocyte count = Retic % x patient’s Hct %
Average normal hct for adult male (45%)
=9X 23%/45% = 4.599%
Calculation of the RPI:
RPI = Corrected retic count in %
Maturation time in days
Maturation
Time
Hematocrit
%
1 day
45
1.5 days
35
2 days
25
3 days
15
RPI= 4.599/ 2
=2.299%
Normal values
Newborn (0-2 weeks):2.5-6.0%
Adult: 0.5-2.0%
27. Which of the following statements characterize a reticulocyte? (Choose ALL of the correct
answers)
More than one answer is correct. Please select all correct answers
a. reticulum visible with new methylene blue N stain
b. pyknotic nucleus still present in the cell
c. contains some organelles used for hemoglobin production
d. reticulum visible with Wright's stain
The correct answers are highlighted below
e. reticulum visible with new methylene blue N stain
f. pyknotic nucleus still present in the cell
g. contains some organelles used for hemoglobin production
h. reticulum visible with Wright's stain
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28. What is the reason for using Perl's Prussian blue stain?

Please select the single best answer
a. enables one to see the Pappenheimer bodies
b. to enhance basophilic stippling
c. confirms the presence of iron containing "granules" in erythrocytes
d. the Prussian blue stain confirms the presence of RNA
29. List 3 conditions in which the reticulocyte count is increased.
a. hemolytic anemia
b. hemorrhage (acute and chronic)
c. in patients with uremia.
d. following treatment of iron-deficiency anemia and the megaloblastic anemias
30. List 3 conditions in which the reticulocyte count is decreased.
A. aplastic anemia
B. aplastic crises of hemolytic anemias
C. ineffective erythropoiesis as seen in thalassemia
D. pernicious anemia
E. sideroblasti
31. List 3 RBC inclusions that could be confused with reticulocytes on a retic smear and state
how you could differentiate them from retics.
a. Howell-Jolly bodies appear as one or sometime two, deep-purple dense structures.
b. Heinz bodies stain a light blue-green and are usually present at the edge of the
erythrocyte.
c. Pappenheimer bodies are more often confused with reticulocytes and are the most
difficult to distinguish. These purple-staining iron deposits generally appear as
several granules in a small cluster. If Pappenheimer bodies are suspected, stain
with Wright-Giemsa to verify their presence.
d. Hemoglobin H inclusions will appear as multiple small dots in every cell.
32. How can you prevent precipitates on the blood film?
Filtration of the stain is necessary when precipitated material is present which can
resemble a reticulocyte
33. What quality control is needed for reticulocyte count?
Three slides should be made for each retic performed. Final calculated reticulocyte
precentage from two slides counts should agree within 15%. If this agreement is not
reached, count the third slide.
 How do you locate the proper area on the blood film to count reticulocytes?
Place the first slide on the microscope stage and, using the low power objective (10x),
find an area in the thin portion of the smear in which the red cells are evenly distributed
and are not touching each other. Carefully change to the oil immersion objective (100x)
and further located an area in which there are approximately 100 red cells per oil
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immersion field.
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EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
Do this by finding a field where the cells are evenly distributed and mentally
divide the field into 4 quadrants. Count the cells in 1 quadrant. If there are
about 25, you are in a good area. There will be a lot of open space between the
red cells.
34. How do you locate the proper area on the blood film to count reticulocytes?




Place the first slide on the microscope stage and, using the low power objective (10x), find
an area in the thin portion of the smear in which the red cells are evenly distributed and
are not touching each other.
Carefully change to the oil immersion objective (100x) and further located an area in which
there are approximately 100 red cells per oil immersion field.
Do this by finding a field where the cells are evenly distributed and mentally divide
the field into 4 quadrants.
Count the cells in 1 quadrant. If there are about 25, you are in a good area. There
will be a lot of open space between the red cells.
35. State the normal range for the following patient populations:
Adult:_______________
Newborn:____________
Normal values:
Adult:
0.5-2.0%
Newborn (0-2 weeks):
2.5-6.0%
36. Calculate the following retic count which was done using the manual method. Show
your calculations.
1st slide = 15 retics counted in 500 RBCs
2nd slide = 17 retics counted in 500 RBCs
1st slide: Retics%= 15X100/500= 3%
2nd slide Retics%= 17X100/500= 3.4%
Average Retics%= 3.2%
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
37. Correct the retic count obtained in the previous question, then calculate RPI. The patient
is an adult female ( Huda Mahmood, 20 yrs old) with a hematocrit of 25%. Show your
calculation.
Patient
name/ID # /
gender/age
Huda
Mahmood, 20
yrs old
Maturation
Time
Hematocrit
%
1 day
45
1.5 days
35
2 days
25
3 days
15
Cells counted
Slide
1:__3%___
Slide2:
__3.4%__
(Slide
3:_____)
Hct
Calculation
Resul
t
25%
Corrected reticulocyte count =
Retic % x patient’s Hct %
Average normal hct female
(42%)
0.95
%
= 3.2X 25%/42% = 1.9%
Normal?
NORMAL
Normal values
for
Calculation of the RPI:
RPI = Corrected retic count in %
Maturation time in days
Newborn:
2.5-6.0%
Adult:
0.5-2.0%
RPI= 1.9/ 2
= 0.95%
Average:_3.2
%
38. Why would a doctor request an RPI?
The reticulocyte count is a means of assessing the erythropoietic activity of the bone
marrow.
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
Reticulocyte Count
An accurate reticulocyte count is key to the initial classification of anemia. Normally,
reticulocytes are red cells that have been recently released from the bone marrow. They are
identified by staining with a supravital dye that precipitates the ribosomal RNA (Fig. 58-12).
These precipitates appear as blue or black punctate spots. This residual RNA is metabolized
over the first 24–36 h of the reticulocyte's lifespan in circulation. Normally, the reticulocyte
count ranges from 1–2% and reflects the daily replacement of 0.8–1.0% of the circulating red
cell population. A reticulocyte count provides a reliable measure of red cell production.
Figure 58-12
Reticulocytes. Methylene blue stain demonstrates residual RNA in newly made red cells.
(From Hillman et al.)
In the initial classification of anemia, the patient's reticulocyte count is compared with the
expected reticulocyte response. In general, if the EPO and erythroid marrow responses to
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
moderate anemia [hemoglobin < 100 g/L (10 g/dL)] are intact, the red cell production rate
increases to two to three times normal within 10 days following the onset of anemia. In the face
of established anemia, a reticulocyte response less than two to three times normal indicates an
inadequate marrow response.
In order to use the reticulocyte count to estimate marrow response, two corrections are
necessary. The first correction adjusts the reticulocyte count based on the reduced number of
circulating red cells. With anemia, the percentage of reticulocytes may be increased while the
absolute number is unchanged. To correct for this effect, the reticulocyte percentage is
multiplied by the ratio of the patient's hemoglobin or hematocrit to the expected
hemoglobin/hematocrit for the age and gender of the patient (Table 58-4). This provides an
estimate of the reticulocyte count corrected for anemia. In order to convert the corrected
reticulocyte count to an index of marrow production, a further correction is required, depending
on whether some of the reticulocytes in circulation have been released from the marrow
prematurely. For this second correction, the peripheral blood smear is examined to see if there
are polychromatophilic macrocytes present. These cells, representing prematurely released
reticulocytes, are referred to as "shift" cells, and the relationship between the degree of shift and
the necessary shift correction factor is shown in Fig. 58-13. The correction is necessary because
these prematurely released cells survive as reticulocytes in circulation for >1 day, thereby
providing a falsely high estimate of daily red cell production. If polychromasia is increased, the
reticulocyte count, already corrected for anemia, should be divided again by a factor of 2 to
account for the prolonged reticulocyte maturation time. The second correction factor varies
from 1–3 depending on the severity of anemia. In general, a correction of 2 is commonly used.
An appropriate correction is shown in Table 58-4. If polychromatophilic cells are not seen on
the blood smear, the second correction is not required. The now doubly corrected reticulocyte
count is the reticulocyte production index, and it provides an estimate of marrow production
relative to normal.
Table 58-4 Calculation of Reticulocyte Production Index
Correction #1 for anemia:
This correction produces the corrected reticulocyte count
In a person whose reticulocyte count is 9%, hemoglobin 7.5 g/dL, hematocrit 23%, the
absolute reticulocyte count = 9 x (7.5/15) [or x (23/45)]= 4.5%
Correction #2 for longer life of prematurely released reticulocytes in the blood:
This correction produces the reticulocyte production index
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
In a person whose reticulocyte count is 9%, hemoglobin 7.5 gm/dL, hematocrit 23%, the
reticulocyte production index
Figure 58-13
Correction of the reticulocyte count. In order to use the reticulocyte count as an indicator of
effective red cell production, the reticulocyte number must be corrected based on the level of
anemia and the circulating life span of the reticulocytes. Erythroid cells take ~4.5 days to
mature. At normal hematocrit levels, they are released to the circulation with ~1 day left as
reticulocytes. However, with different levels of anemia, erythroid cells are released from the
marrow prematurely. Most patients come to clinical attention with hematocrits in the mid-20s
and thus a correction factor of 2 is commonly used because the observed reticulocytes will live
for 2 days in the circulation before losing their RNA.
Premature release of reticulocytes is normally due to increased EPO stimulation. However, if
the integrity of the bone marrow release process is lost through tumor infiltration, fibrosis, or
other disorders, the appearance of nucleated red cells or polychromatophilic macrocytes should
still invoke the second reticulocyte correction. The shift correction should always be applied to
a patient with anemia and a very high reticulocyte count to provide a true index of effective red
cell production. Patients with severe chronic hemolytic anemia may increase red cell production
as much as six- to sevenfold. This measure alone, therefore, confirms the fact that the patient
has an appropriate EPO response, a normally functioning bone marrow, and sufficient iron
available to meet the demands for new red cell formation. Table 58-5 demonstrates the normal
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
marrow response to anemia. If the reticulocyte production index is <2 in the face of established
anemia, a defect in erythroid marrow proliferation or maturation must be present.
Table 58-5 Normal Marrow Response to Anemia
Hematocrit Production Index Reticulocytes (incl corrections) Marrow M:E Ratio
45
1
1
3:1
35
2.0–3.0
4.8%/3.8/2.5
2:1–1:1
25
3.0–5.0
14%/8/4.0
1:1–1:2
15
3.0–5.0
30%/10/4.0
1:1–1:2
Shift Correction
Estimating RBC production by using the corrected reticulocyte count may yield
erroneously high values in patients when there is a premature release of younger
reticulocytes, “stress or shift” reticulocytes, from the marrow (owing to increased
erythropoietin stimulation). The maturation time of these reticulocytes in the bone
marrow is shortened, while the maturation time in the peripheral blood is lengthened.
Therefore the number of reticulocytes in the perpheral blood may be increased without
any corresponding increase in erythropoietic activity.
Shift Correction should be considered during periods of intense erytherpoietic stress
when stress macroreticulocytes are relaesed in the circulation prematurely.
A shift correction was proposed to compensate for increased duration in the circulating
blood. This correction is based on the haematocrit. This maturation time is as follows:
Hematocrit%
Peripheral Blood Maturation
Time (Days)
45
1 day
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
35
1.5 days
25
2 days
15
3 days
The peripheral blood smear should be reviewed carefully for the presence of many
polychromatophilic macrocytes, thus indicating stress reticulocytes and the need for
correction for both the RBC count and the presence of stress reticulocytes.
Reticulocyte Production Index (RPI)
A reticulocyte count corrected for both hematocrit and shift is known as the reticulocyte
production index (RPI).
Calculation of the RPI:
RPI = Corrected retic count in %
Maturation time in days


RPI equal to or greater than 3 represents an adequate response to anemia by
the bone marrow
RPI of less than 2 is considered an inadequate response of erythopoiesis by the
bone marrow to a state of anemia.
http://www.benhhoc.com/index.php?do=viewarticle&artid=1828&title=chapter-058-anemiaand-polycythemia
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
Figure e11-2. Reticulocyte count preparation. This new methylene blue–stained blood...
Harrison's Online > Chapter e11. Atlas of Hematology and Analysis of Peripheral Blood
Smears > Atlas of Hematology and Analysis of Peripheral Blood Smears > Acknowledgment
http://accessmedicine.net/search/searchAMResultImg.aspx?searchStr=leukocyte+count&search
Type=2&fullTextStr=leukocyte+count&resourceID=4&narrowing=yes
blood flim from a cat LEFT wrights stain, RIGHT brilliant cresyl blue stain.
descibe the abormalities
Reticulocytes
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count






MLAB 1315 Hematology
Although the nucleus has been extruded, the reticulocyte is still considered immature
because it retains numerous organelles needed for hemoglobin production, such as
ribosomes, mitochondria, and fragments of the Golgi apparatus.
The reticulocyte is slightly larger (10 microns) than the mature erythrocyte.
A reticulocyte normally remains in the bone marrow for one or two days before
entering the circulation and its final 24 hours of maturation. The red cell is mature
when hemoglobin production is complete and the organelles have disintegrated.
Reticulocytes normally make up 0.5 - 1.5% of the peripheral blood red cells.
They appear blue/gray on the Wright's stained smear. The residual RNA in the
cytoplasm causes the blue/gray color.
The terms, polychromasia or polychromatophilic, are used to describe these cells on
a Wright's stained preparation as seen on the left in the image. A supravital stain
such as new methylene blue N or brilliant cresyl blue is used to stain reticulocytes
for an actual count as seen on the right in the image.
STUDY QUESTIONS
Name_ _______________________________
Date_________________________________
1 pt
39.
What is the reticulocyte used to assess?
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
MLAB 1315 Hematology
2 pt
40.
What criterion is used to identify reticulocytes?
1 pt
41.
What is reticulum composed of?
1 pt
42.
1 pt
43.
What is a “stress reticulocyte”?
3 pt
44.
List 3 conditions in which the reticulocyte count is increased.
A.
B.
C.
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
3 pt
45.
MLAB 1315 Hematology
List 3 conditions in which the reticulocyte count is decreased.
A.
B.
C.
6 pt
6 pts. 9. List 3 RBC inclusions that could be confused with reticulocytes on a retic
smear and state how you could differentiate them from retics.
A.
B.
C.
1 pt
10 How can you distinguish between reticulum and artifact?
2 pt
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LAB EXERCISES
MLAB 1315
EXERCISE 3: Reticulocyte Count
11.
State
the
normal
range
for the
followi
ng
patient
populat
ions:
2 pt
MLAB 1315 Hematology
Adult:_______________
Newborn:____________
12. Calculate the following retic count which was done using the manual method.
Show your calculations.
1st slide = 15 retics counted in 500 RBCs
2nd slide = 17 retics counted in 500 RBCs
1 pt
13. Correct the retic count obtained in question # 12. The patient is an adult female
with a hematocrit of 25%. Show your calculation.
2 pts
14. Calculate the following retic count which was done using the Miller disc method.
The patient is an adult male. Show your calculation.
# retics counted in larger square: 26
# RBCs in smaller square: 111
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LAB EXERCISES
MLAB 1315
[Type text]
2 pts
88. if there were 18 reticulocytes per 1000 red cells, what will be the percentage of
reticulocytes ? what will be the absolute reticulocyte count if the RBC count is 4.5 x
106/ul?
retics % is: 18/1000 x 100% = 1.8%
If the RBC = 4.5 x 106/ul Absolute reticulocyte count
=
1.8 x 4.5 x 106 =
100
81 x 103/ul
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LAB EXERCISES
MLAB 1315
[Type text]
15. Why would a doctor request an RPI?
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LAB EXERCISES
MLAB 1315